Regeneration and plastid transformation approaches in Arabidopsis thaliana and Rapid-Cycling

Regeneration and plastid transformation approaches in Arabidopsis thaliana and

Rapid-Cycling Brassica rapa

Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften der Fakultät für Biologie der

Ludwig-Maximilians-Universität München

Vorgelegt von Areli Herrera Díaz

aus Mexiko

München, 2011

1. Gutachter: Prof. Dr. Hans-Ulrich Koop

2. Gutachter: Prof. Dr. Jörg Nickelsen

Tag der mündlichen Prüfung: 22. Juli 2011

Contents

1

CONTENTS

1 ZUSAMMENFASSUNG ............................................................................ 3

2 SUMMARY .......................................................................................... 5

3 INTRODUCTION .................................................................................. 7

3.1 Plastids ...................................................................................... 7

3.1.1 Plastid division ........................................................................ 8

3.1.2 Plastid inheritance ................................................................... 8

3.1.3 Plastid genome ...................................................................... 10

3.1.4 Plastid gene expression .......................................................... 11

3.2 Genetic transformation of plastids in higher plants ................. 12

3.2.1 Gene transfer methods and transformation vectors .................... 12

3.2.2 Selection markers .................................................................. 13

3.2.3 Reporter genes ...................................................................... 16

3.2.4 Plastid transformation in Nicotiana tabacum .............................. 16

3.2.5 Plastid transformation in different species ................................. 18

3.3 Arabidopsis thaliana as model organism ................................. 18

3.3.1 Tissue culture in Arabidopsis thaliana ....................................... 20

3.3.2 Protoplast culture in Arabidopsis thaliana .................................. 21

3.4 Rapid-Cycling Brassica rapa, a new model organism ............... 22

4 RESEARCH AIMS ............................................................................... 24

5 MATERIALS AND METHODS ................................................................. 25

5.1 Chemicals and enzymes ........................................................... 25

5.2 Kits .......................................................................................... 27

5.3 Disposable material ................................................................. 27

5.4 Equipment and instruments ..................................................... 27

5.5 Software and internet tools ..................................................... 28

5.6 Biological material ................................................................... 28

5.6.1 Plant material ....................................................................... 28

5.6.2 Medium for culture of bacteria ................................................. 28

5.7 Molecular methods .................................................................. 29

5.7.1 Isolation of genomic DNA of plants .......................................... 29

5.7.2 Isolation of plasmid DNA ........................................................ 30

5.7.3 Gel electrophoresis ................................................................ 30

5.7.4 DNA purification and concentration .......................................... 31

5.7.5 DNA quantification ................................................................. 31

5.7.6 DNA restriction and modification .............................................. 32

5.7.7 DNA ligation .......................................................................... 32

5.7.8 Preparation of competent cells ................................................ 33

5.7.9 Heat shock transformation of Escherichia coli ............................ 33

5.7.10 PCR and sequencing ............................................................ 34

5.7.11 Southern blot hybridization .................................................. 35

5.8 Plant tissue culture .................................................................. 38

5.8.1 Shoot induction from seeds and cotyledons in A. thaliana ........... 39

5.8.2 Protoplast isolation, embedding and culture in A.thaliana............ 41

Contents

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5.8.3 Plant tissue culture in Rapid-Cycling Brassica rapa ..................... 43

5.8.4 Plant tissue and protoplast culture in Nicotiana tabacum ............ 45

5.9 Plant transformation ............................................................... 46

5.9.1 PEG treatment of protoplasts .................................................. 46

5.9.2 DNA transfer using the particle gun method .............................. 47

5.10 Selection conditions ................................................................ 49

5.11 Microscopy .............................................................................. 49

6 RESULTS ......................................................................................... 50

6.1 Towards plastid transformation in Arabidopsis thaliana .......... 50

6.1.1 Regeneration from cotyledons in A. thaliana .............................. 50

6.1.2 Regeneration of A. thaliana from seed explants ......................... 52

6.1.3 A. thaliana protoplast culture and regeneration ......................... 56

6.1.4 Evaluation of the sensitivity of A. thaliana protoplast derived

colonies to selection agents ................................................................. 60

6.1.5 Construction of A. thaliana plastid transformation vectors ........... 61

6.1.6 Particle gun and PEG-based plastid transformation experiments in

A. thaliana ........................................................................................ 69

6.2 Towards plastid transformation in Rapid-Cycling B. rapa ........ 75

6.2.1 Plant regeneration from Rapid-Cycling Brassica rapa .................. 75

6.2.2 Particle gun experiments for plastid transformation in Rapid-Cycling

Brassica rapa ..................................................................................... 77

6.3 AmCyan as visual marker for plastid transformation in Nicotiana

tabacum ............................................................................................. 80

7 DISCUSSION .................................................................................... 85

7.1 Towards plastid transformation in Arabidopsis thaliana .......... 85

7.1.1 Regeneration protocols in A. thaliana ....................................... 85

7.1.2 Species specific vectors in A. thaliana ...................................... 89

7.1.3 Particle gun-mediated plastid transformation ............................ 93

7.1.4 PEG-mediated plastid transformation ....................................... 96

7.2 Towards plastid transformation in Rapid-Cycling B. rapa ........ 97

7.3 Nicotiana tabacum, a positive control in plastid transformation ..

................................................................................................ 98

7.3.1 Plastid transformation in A. thaliana and Nicotiana tabacum ...... 100

7.4 Future directions in plastid transformation of A. thaliana ...... 101

8 ABBREVIATIONS ............................................................................. 103

9 REFERENCES .................................................................................. 104

10 CURRICULUM VITAE ......................................................................... 122

11 ACKNOWLEDGEMENTS ...................................................................... 123

12 ERKLÄRUNG ................................................................................... 124

Zusammenfassung

3

1 Zusammenfassung

Plastidentransformation ist eine wertvolle Methode in der Biotechnologie,

weil sie hohe Expressionsraten rekombinanter Proteine ermöglicht und weil die

Transgene nicht durch Pollen verbreitet werden können. In der

Grundlagenforschung können die Funktion und Regulation von plastidären

Genen mit Hilfe der Plastidentransformation näher untersucht werden.

Diese Studie konzentriert sich auf die drei wichtigsten Schritte bei der

Plastidentransformation in Arabidopsis thaliana und in Rapid-Cycling Brassica rapa

(RCBr); die Etablierung eines Regenerationsprotokolls, die Konstruktion von

artspezifischen Vektoren und die Verwendung unterschiedlicher Transfor-

mationsprotokolle.

Um die Erzeugung von fertilen Pflanzen aus transformierten Zellen zu

ermöglichen, wurde zuerst ein zuverlässiges Regenerationsprotokoll etabliert.

Fertile Pflanzen von Arabidopsis thaliana wurden mit hoher Regenerationsrate aus

Kotyledonen, Kalli von Samen und Protoplasten regeneriert. In RCBr konnten

fertile Pflanzen aus Gewebekulturen von Hypokotylen regeneriert werden.

Für die Übertragung von Genen in das Genom der Plastiden wurden geeignete

Vektoren mit verschiedenen selektiven und visuellen Markern konstruiert. Diese

sorgen für eine ortsspezifische Integration der gewünschten Sequenzen und

ermöglichen die Selektion der transformierten Zelllinien. In dieser Studie wurden

artspezifische Vektoren für Nicotiana tabacum, Arabidopsis thaliana und RCBr

konstruiert und kloniert, die Aminoglykosid-Resistenz-Marker wie das aadA-Gen

(Resistenz gegen Spectinomycin und Streptomycin), das nptII- oder das aphA6-Gen

(Resistenz gegen Kanamycin) enthielten. Um mit den Herbiziden

Phosphinothricin oder Bialaphos selektieren zu können, wurde das bar-Gen

eingeführt. Darüber hinaus wurden Fluoreszenz-Marker wie GFP, DsRed und

AmCyan als visuelle Marker hergenommen.

Nach der Etablierung eines Regenerationsprotokolls und der Konstruktion

spezifischer Vektoren wurden zwei unterschiedliche Protokolle für die

Plastidentransformation, die Particle Gun- oder die PEG (Polyethylenglykol)-

Zusammenfassung

4

vermittelte Transformationsmethode, angewandt. Allerdings konnten bisher keine

transformierten Pflanzen von Arabidopsis thaliana und RCBr erhalten werden. Dass

das Transformationsprotokoll grundsätzlich funktioniert, konnte durch die

Verwendung von Nicotiana tabacum gezeigt werden. Plastiden von Nicotiana

tabacum wurden erfolgreich mit beiden Transformationsmethoden transformiert,

und AmCyan konnte als neuer visueller Marker etabliert werden. Bei diesen

Experimenten konnten fertile, homoplasmatische Tabakpflanzen erhalten werden,

was durch Southern-Blot-Analyse und reziproke Kreuzungen bestätigt wurde.

Somit konnte die Funktionalität der verwendeten transgenen Expressions-

kassetten eindeutig nachgewiesen werden.

Summary

5

2 Summary

Plastid transformation is a well-established tool for biotechnology as it allows high

expression levels of proteins and as it provides biological transgene containment

because of maternal inheritance of cytoplasmic genes in most crops. In basic

research the function and regulation of plastid encoded genes can be further

studied with the help of plastid transformation.

This study is focused on the three most critical steps to achieve plastid

transformation in Arabidopsis thaliana and Rapid-Cycling Brassica rapa (RCBr), the

establishment of a regeneration protocol, the construction of species specific

vectors and the use of different transformation protocols.

First, to allow the generation of fertile plants out of transformed cells, a reliable

regeneration protocol was established. Fertile plants of Arabidopsis thaliana were

obtained with high regeneration efficiencies from cotyledons, seed derived calli

and protoplasts. In RCBr, fertile plants could be regenerated from tissue culture of

hypocotyls.

For introduction of genes into plastids, appropriate vectors with different selection

and visual markers were constructed. These ensure site specific integration of

desired sequences and enable selection of transformed cell lines. In this study,

species specific vectors for Nicotiana tabacum, Arabidopsis thaliana and RCBr were

designed and cloned, using aminoglycoside resistance markers such as the aadA

gene (confers resistance to spectinomycin and streptinomycin), the nptII or the

aphA6 gene (conferring resistance to kanamycin). To allow the selection through

the herbicides phosphinothricin or bialaphos, the bar gene was employed. In

addition, fluorescence markers such as GFP, DsRed and AmCyan were taken as

visual markers.

After the establishment of a regeneration protocol and the construction of specific

vectors, two different protocols for plastid transformation, the particle gun- or

PEG (Polyethylene glycol)-mediated transformation method, were applied.

However, no transformed plant in Arabidopsis thaliana and RCBr could be

Summary

6

recovered so far. That the transformation protocol does work in principal could be

shown by the use of Nicotiana tabacum. Plastids of Nicotiana tabacum were

successfully transformed using both transformation methods and AmCyan as new

visual marker. Out of these experiments, fertile homoplasmic tobacco plants could

be obtained as was shown by Southern blot analysis and reciprocal crosses. Thus,

the functionality of the used transgene expression cassettes was clearly proven.

Introduction

7

3 Introduction

3.1 Plastids

Plastids are the defining feature in plants and algae. According to the

endosymbiosis theory, plastids were free-living cyanobacteria over one billion

years ago, before becoming endosymbionts.

Plastids are double-membrane organelles developed from undifferentiated

proplastids in dividing meristematic cells (Whatley, 1978). Proplastids

differentiate into several plastid types like chloroplasts (chlorophyll),

chromoplasts (carotenoids), amyloplasts (starch), elaioplasts (lipids), leucoplasts

(white plastids), etioplasts (plastids in the complete absence of light), and

gerontoplasts (plastids found in senescing) (Keskitalo et al., 2005). All plastid types

can be interconverted, except the proplastids (Wise, 2006).

The chloroplasts are the most common form of plastids in plants (Figure 3.1). They

are able to convert light into chemical energy through a process called

photosynthesis. Chloroplasts are not just involved in photosynthesis, but they also

play an essential role in plant metabolism (Neuhaus and Emes, 2000). Plastids

produce fatty acids, aromatic and non-aromatic amino acids, purine and

pyrimidine bases, isoprenoids (carotenoids and sterols) and tetrapyrroles

(chlorophyll) (Neuhaus and Emes, 2000; Lopez-Juez and Pyke, 2005; Waters and

Langdale, 2009).

Figure 3.1 Chloroplast. (a) Electron micrograph of a chloroplast in a leaf of grass, showing the highly folded system of internal membranes containing the chlorophyll molecules by which light is absorbed. (b) An illustration of (a) Modified from Alberts et al., 2002.

Introduction

8

3.1.1 Plastid division

Plastid proliferation occurs by binary fission of pre-existing plastids.

Plastids can only be transmitted to daughter cells through cell division; de novo

synthesis of plastids is not known (Maple and Møller, 2007).

Plastid division starts with the formation of a ring like structure in the middle of

the plastid, known as plastid division (PD) ring. This structure can be easily

observed with the light microscope and two PD rings could be observed using

transmission electron microscopy (TEM) (Figure 3.2). One PD ring is located on

the stromal face of the chloroplast's inner membrane (bacterial origin) and the

other one is located on the cytosolic face of the outer membrane (cytosolic division

machinery). A number of proteins involved in plastid division have been

identified and characterized, for example FtsZ, MinD, MinE and ARTEMIS.

However, the way of how all these proteins interact with each other to coordinate

plastid division remains unknown (Fulgosi et al., 2002; López-Juez, 2007; Maple

and Møller, 2007).

3.1.2 Plastid inheritance

The inheritance of genes from organelles like plastids and mitochondria in

plants, unlike the nucleus, does not follow Mendel´s laws (Birky, 2001) (Figure

3.3). Plastid inheritance can be studied in naturally variegated plants; initial

evidences of an extra nuclear non-Mendelian inheritance of variegation were

described in Mirabilis jalapa (Correns, 1909) and Pelargonium zonale (Baur, 1909).

Figure 3.2 Dividing plastids (a) Nomarski optic micrographs showing accumulation of shaped pre-division plastids caused by keeping spinach leaf discs under low light intensity for 5 d. (b) Transmission electron micrograph showing constriction prior to division (scale bar = 1 μm). (c) Diagram of constricting doublet ring visible as densely stained areas inside and outside double plastid membrane. From: http://plantsinaction.science.uq.edu.au

Introduction

9

The plastid inheritance theory and the random sorting out of plastids was

proposed by Erwin Baur using the term “chromatophores” for plastids (Baur,

1909; Hagemann, 2000; 2010).

Plastid inheritance in higher plants can be either uniparental or biparental. While

paternal plastid inheritance can be found in conifers (Szmidt et al., 1987),

biparental inheritance could be described only in some species like Pelargonium,

Oenothera and Hypericum (Hagemann and Schröder, 1989).

In angiosperms, plastids are mainly maternally inherited (Figure 3.3), including

Beta, Mirabilis, Nicotiana, Zea, Arabidopsis thaliana and many others. In Arabidopsis

thaliana and Nicotiana tabacum a low frequency of paternal plastids in the seeds

progeny have been reported (Hagemann and Schröder, 1989; Huang et al., 2003;

Azhagiri and Maliga, 2007).

Figure 3.3 Maternal inheritance of plastids. In reciprocal crosses between green and white plants (or branches) of Mirabilis jalapa, the plastids of the mother plant determines the type of progeny. The cross white (female) x green (male) gives only white Fl-seedlings; the cross green (female) x white (male) gives only green F1- seedlings. A variegated mother plant (crossed with a green or white mate) can produce variegated, white and green seedling. Modified from Griffiths et al., 1999.

Using different cytological and molecular methods, the routes for maternal

inheritance could be analyzed. For example: physical exclusion of the plastids

during pollen mitosis, elimination of plastids by formation of enucleated

cytoplasmic bodies and autophagic degradation of male plastids in different

stages (Nagata, 2010).

The sorting out of plastomes is due to a segregation process upon organelle

division as well as random organelle segregation upon cell division. The first

model of plastid inheritance was developed using the plant Epilobium. This

inheritance model of partitioning uses the mathematical model of sampling

Introduction

10

without replacement and the hypergeometric probability distribution (Michaelis,

1962a). Although the model is not exactly applicable, it helped to predict that

vegetative segregation can be completed within one plant generation depending

on the number of observed proplastids in the plant embryo cell (Michaelis, 1962b;

Birky, 2001).

3.1.3 Plastid genome

The genetic information in plants is distributed among three cellular

compartments: the nucleus, the mitochondria and the plastids. The plastids have

their own genome, known as plastome. Plastomes from land plants are circular

molecules of double stranded DNA, between 120 and 180 kb in size (Sugiura,

2003). However, different evidences described structural plasticity of the plastome

in Arabidopsis thaliana and Nicotiana tabacum, which are characterized by the

presence of circular, linear, bubble D/loop, lasso-like and unclassified plastome

structures (Bendich and Smith, 1990; Lilly et al., 2001; Bendich, 2004; Scharff and

Koop, 2006).

Interestingly, plastids are highly polyploid and their plastomes are organized as

nucleoids which are attached to the inner membrane (Sato et al., 1997). A single

leaf cell may contain hundreds of chloroplasts, each containing nucleoids with

several copies of the plastome (Thomas and Rose, 1983). In average the number of

copies of plastomes per leaf cell ranges from 1000 to 1700 in Arabidopsis thaliana

and reaches up to 50,000 in wheat (Bendich, 1987; Zoschke et al., 2007). Generally,

the plastome of higher plants is conserved in size, organization and sequence.

Plastome contains an inverted repeat region A and B (IRA and IRB respectively)

and two single unique regions known as small single copy (SSC) and large single

copy (LSC) regions (Sato et al., 1999; Sugiura, 2003). Exceptions to this rule are

some legumes and conifers (Wakasugi et al., 2001). During the last 15 years, more

than 130 complete plastomes have been sequenced and deposited in the GenBank

Organelle Genome Resource (Yang et al., 2010).

Chloroplast DNA is replicated transcribed and translated independently within

the cell. However, chloroplasts strongly depend on imported proteins that are

Introduction

11

encoded in the nucleus and imported into the organelle. For Arabidopsis thaliana it

has been proposed that the nucleus encodes for about 2100 chloroplast proteins. In

contrast, the whole chloroplast genome encodes for 117 proteins as well as tRNAs

and rRNAs (Richly and Leister, 2004; López-Juez, 2007).

3.1.4 Plastid gene expression

Chloroplast gene organization, transcription and translation is similar to

that of eubacteria. Chloroplast genes are organized mainly in operons and can be

expressed as polycistronic units (Sugiura, 1992).

Transcription in plastids is mainly executed by two or three DNA-dependent RNA

polymerases. The plastid genome encodes for one RNA polymerase (PEP)

consisting of four core subunits rpoA, rpoB, rpoC1 and rpoC2. Interestingly, PEP

recognizes similar promoters to the E. coli sigma70 –type, including the -10

(TATAAAT) and the -35 (TTGACA) consensus region (Hager and Bock, 2000).

Several RNA polymerases (NEP) like RPOTp and RPOTpm are imported from the

nucleus into plastids (Krause et al., 2000; Zerges, 2000; Courtois et al., 2007).

Once a gene or polycistron has been transcribed, it may be modified post-

transcriptionally, including RNA editing (e.g. base change from C to U) (Bock,

1998), RNA splicing (Bock and Maliga, 1995), 5ánd 3´-end maturation by

ribonucleases (Monde et al., 2000b) and cleavage of the polycistronic RNA chains.

Different evidence showed that the 5ánd 3úntranslated region (UTRs) of plastid

RNA´s are important for the stability of mRNA (Eibl, 1999; Monde et al., 2000a;

Zou et al., 2003).

In the same way as in bacteria, chloroplast translation utilizes 70S ribosomes

which do not require 5ćaps or 3´poly (A) tails. However, the Shine-Dalgarno

(SD)-like sequence required for ribosomal recognition and translation in bacteria,

is not found in many chloroplasts´ mRNAs (Sugiura et al., 1998).

The regulatory mechanisms of gene expression in plastids at the translational and

posttranslational level remain unknown. Recently, Apel et al., suggested that the

major determinants for the protein stability in plastids are located in the N-

Introduction

12

terminal region. Using mutated N-terminal regions they showed that a GFP-

protein harboring glutamate or methionine in the second position was strongly

accumulated in plastids (Apel et al., 2010).

In summary, the level of protein expression in plastids not only depends on the

transcription level and mRNA-stability, it also depends on protein stability.

3.2 Genetic transformation of plastids in higher plants

Due to the high level of gene expression, plastids are an attractive

compartment for the expression of recombinant proteins. Recently, the expression

level of a phage-lytic protein in chloroplasts reached 70 % of the plant total soluble

protein fraction (Oey et al., 2008). Another advantage is the expression of several

genes in a single operon in chloroplasts (Lossl et al., 2003; Quesada-Vargas et al.,

2005). Additionally, gene silencing and position effects on transgenes cannot be

observed in plastids. From a biosafety perspective, plastid transformation

significantly increases transgene containment because plastids are maternally

inherited in most plants (Figure 3.3) (Bogorad, 2000; Maliga, 2003; Hagemann,

2010). Taken all together, these are the major advantages of this technology.

Stable plastid transformation was first achieved in the alga Chlamydomonas

reinhardtii (Boynton et al., 1988; Blowers et al., 1989). Almost 20 years ago, it was

also possible to introduce DNA into tobacco chloroplasts for the first time through

the particle gun method (Svab et al., 1990). Currently, plastid transformation

represents a highly active research area which is documented in more than 25

reviews (e.g. Bogorad, 2000; Bock, 2001; Daniell, 2002; Bock and Khan, 2004;

Maliga, 2004; Koop et al., 2007; Maliga and Bock, 2011).

3.2.1 Gene transfer methods and transformation vectors

In general, there are two DNA delivery methods for a stable introduction of

foreign DNA into plastids. The first one consists in bombarding of tissue or cells

with DNA coated particles (Daniell et al., 1990; Svab et al., 1990). The second

method treats isolated protoplasts (plant cells without cell wall) with polyethylene

Introduction

13

glycol (PEG). PEG induces pores in cell membranes allowing the entrance of DNA

into plastids, however, the exact mechanism is still unknown (Golds et al., 1993;

O'Neill et al., 1993).

A prerequisite for the expression of heterologous proteins in chloroplasts is that

the gene of interest must be flanked by plastid regulatory elements: a promoter

with a 5´ untranslated region (UTR) and a ribosome binding sequence (e.g. Shine-

Dalgarno sequence). A frequent combination used a plasmid containing the strong

constitutive plastid 16 S ribosomal RNA promoter (Prrn) in combination with the

viral T7g10-5´UTR (Staub et al., 2000) or the psbA control elements (promoter, 5´-

UTR) (Fernandez-San Millan et al., 2003). A 3´ UTR region generally includes a

stem-loop structure to stabilize the transcript. The most often used 3´UTRs are

derived from the psbA, rbcL and rps16 genes (Koop et al., 2007).

The genetic transformation of plastids follows the principles of homologous

recombination. Therefore, the transgene has to be flanked on either side by

plastome sequences ensuring the integration of the transgene into a specific region

within the plastome (Svab et al., 1990) (Figure 3.4). Both sequences are required to

allow the integration of foreign genes within the plastome. In average homologous

flanks have a size of 1 kpb (Maliga, 2004). Stable integration of the transgene has

been achieved in several regions of the plastome. Therefore intergenic regions are

often chosen: rbcL-aacD (Svab and Maliga, 1993); trnV-rps12/7 (McBride et al.,

1995); trnI-trnA (Kang et al., 2003); trnN-trnR (Zou et al., 2003) and trnfM-trnG

(Zhou et al., 2007). It is also recommended that only species specific vectors should

be used for plastid transformation (Verma and Daniell, 2007).

3.2.2 Selection markers

Successful plastid transformation requires selection markers. The first

selection marker described for plastid transformation in higher plants was a

mutated plastid 16 S ribosomal RNA gene. This mutation confers resistance to

spectinomycin (Svab et al., 1990; Staub and Maliga, 1992). The most common

selection marker used for plastid transformation is the bacterial spectinomycin

Introduction

14

resistance gene aadA encoding for a 3áminoglycoside-adenyltransferase (AADA).

This protein inactivates spectinomycin and streptomycin antibiotics (Svab and

Maliga, 1993). Figure 3.4 shows the standard components of a plastid

transformation vector.

Another selection marker is the neomycin phosphotransferase gene (nptII) which

confers resistance to kanamycin. This gene is generally used in nuclear

transformation because in plastid transformation it was not that efficient (Maliga,

2003). However, after the introduction of a Prrn promoter and a fusion of the first

nucleotides of the gfp gene allowed a higher expression of NPTII in chloroplasts

and thus the successful selection of transplastomic plants (Scharff and Koop, 2007).

In 2002, the aminoglycoside phosphotransferase VI gene aphA6 from Acinetobacter

baumannii was described as a resistance marker for plastid transformation in

tobacco. This aphA6 gene encodes for kanamycin resistance (Bateman and Purton,

2000; Huang et al., 2002).

Another version of selection markers combines aminoglycoside selection with

secondary selection genes as bar and pat genes which encode for phosphinothricin

acetyl transferases that confer resistance to the herbicides bialaphos and

phosphinotricin. Additionally the epsps gene (5-enolpyruvylshikimate-3-

Figure 3.4 Targeting a transgene expression cassette into the chloroplast genome. The expression cassette consists of the aadA gene coding sequence fused to a chloroplast promoter (P) and 5´-untranslated region (5ÚTR) on its 5´side and to a chloroplasts 3´-untranslated region (3ÚTR) on its 3´side. Upon cell transformation with the plasmid, in which the expression cassette is flanked by chloroplast DNA sequences corresponding to the targeting region (indicated by a thick line), the cassette is inserted into the chloroplasts genome by homologous recombination. Modified from Rochaix, 1997.

Introduction

15

Figure 3.5 Plastid transformation. (a) Tobacco leafs are bombarded. Resistant green shoots appear on regeneration medium with antibiotic after 4-12 weeks. Shoots are usually heteroplasmic and at least three rounds of selection are needed to obtain homoplasmy (each round: 4 weeks). Homoplastomic lines are identified by Southern blotting then rooted in a free hormone medium. (b) Schematic description of sorting out of transformed chloroplasts. After bombardment just one or few chloroplasts are transformed (red color). Subsequent cell and organelle divisions in the presence of selective pressure favor the multiplication of transformed chloroplasts and the elimination of wild type cells. (c) Schematic description of the generation of homoplasmic chloroplasts. The target region is indicated as an open box. After the biolistic experiments, the resistance cassette (red) is integrated into the chloroplast genome. In the heteroplasmic state (left side) both wild type and transformed copies are present. After several rounds of selection, the homoplasmic chloroplasts are obtained.

phosphate synthase) encodes for a resistance to glyphosate based herbicides

(Iamtham and Day, 2000; Ye et al., 2003).

After the bombardment of foreign DNA into the cell, it can be assumed that at the

most one or a few plastomes are transformed. High selective pressure is needed to

allow the elimination of the wild type plastomes and the amplification of

transformed plastomes. Homoplasmic or homoplastomic cells (uniform

population of transformed plastids) are obtained through several rounds of cell

division and organelle segregation. Thus, homoplasmy can be achieved through

several rounds of regeneration starting from leaves or tissue explants on selection

medium (Svab and Maliga, 1993) (Figure 3.5).

Introduction

16

3.2.3 Reporter genes

The use of reporter genes in plastid transformation allows the

determination of transformation efficiency. One of the first reporter genes used in

plastid transformation was the gus reporter gene (β-glucuronidase). Gus activity

can be visualized by histochemical staining and fluorescence assays. Another

reporter gene is cat (chloramphenicol acetyl transferase), first described in 1990, it

took 20 years to be reported as a stable reporter gene (Daniell et al., 1990; Zou et al.,

2003; Li et al., 2010).

The use of fluorescent proteins such as GFP (green fluorescent protein) facilitates

the selection of transformants in living cells as has been demonstrated for transient

experiments (Hibberd et al., 1998). Fusion between the aadA and gfp genes have

been used as a visual and selection marker in Nicotiana tabacum (Khan and Maliga,

1999). Moreover, GFP has been also used as visual marker in lettuce, potato,

Lesquerella, rice and poplar (Skarjinskaia et al., 2003; Lelivelt et al., 2005; Lee et al.,

2006; Okumura et al., 2006; Valkov et al., 2010). Last but not least, other fluorescent

markers have been employed, such as the synthetic red shift variant of GFP (Reed

et al., 2001) or the YFP (Yellow fluorescent protein) (Buhot et al., 2006; Zhou et al.,

2007). Recently, the bacterial luciferase operon, known as lux operon was

expressed in tobacco plants. The light emission can be detected even with the

naked eye in plastid transformed plants (Krichevsky et al., 2010). Non-lethal visual

markers represent a good alternative for the characterization of transformed

plants.

3.2.4 Plastid transformation in Nicotiana tabacum

Tobacco is the best higher plant model for plastid transformation. The use

of plastid transformation in tobacco helped to elucidate the function of genes as

well as the transcription and translation machinery in plastids. For example, DNA

replication, transcription and RNA processing, was further analyzed in this model

including regulatory elements and the stability of proteins (Bock et al., 1993; Bock

and Maliga, 1995; Muhlbauer et al., 2002; Zou et al., 2003; Oey et al., 2008).

Introduction

17

Table 3.1 Expression of several proteins in Nicotiana tabacum

Furthermore, more than 30 plastid genes have been inactivated in tobacco for

functional analysis (Koop et al., 2007). Additionally, more than 50 different

recombinant proteins of biotechnological use have been expressed in tobacco

chloroplasts. This includes plants which are resistant to herbicides, insects and

diseases (McBride et al., 1995; DeGray et al., 2001; Lutz et al., 2001). On top of that,

several biopharmaceutical proteins and several vaccine antigens have been

expressed in tobacco plastids (e.g. Daniell et al., 2005; Bock, 2007; Koop et al., 2007).

A summary of proteins expressed in tobacco is shown in Table 3.1.

Genes Protein or final product Reference

reporter genes

uidA β- Glucuronidase (Staub and Maliga, 1993)

neo neomycin phosphotransferase (Carrer et al., 1993)

cat chloranphenicol acetyl transferase (Li et al., 2010)

gfp green fluorescent protein (Sidorov et al., 1999)

e-yfp e-yellow fluorescent protein (Buhot et al., 2006)

herbicide resistance

bar phosphinothricin acetyl transferase (Lutz et al., 2001)

epsps 5-enolpyruvylshikimate-3-phosphate synthase (Daniell et al., 1998)

insect resistance

cry1Ac Bacillus turingencis toxin (McBride et al., 1995)

cry2Aa2 operon Bacillus turingencis toxin (De Cosa et al., 2001)

bioplastics

egl12I PBP (GVGVP) (Protein-based polymer) (Guda et al., 2000)

phbC-phbA-phbB PHB (Polyhydroxybutyrate) (Lossl et al., 2005)

pathogen resistance

msi-99 antimicrobial peptide (DeGray et al., 2001)

Protegrin-1 antimicrobial peptide (Lee et al., 2011)

drought or salt tolerance

tpsI threalose-6- phosphate synthase (Lee et al., 2003)

badh betaine aldehyde dehydrogenase (Daniell et al., 2001)

biopharmaceuticals

hgh somatotropin (Staub et al., 2000)

igf human insulin like growth factor (Daniell et al., 2009)

ifn-alpha2b interferon alpha 2b (Arlen et al., 2007)

vaccine in plants

tetC Tetanus toxin C (Clostridium tetani) (Tregoning et al., 2003)

ltb B subunit (E. coli enterotoxigenic) (Kang et al., 2003)

pag PA protective antigen anthrax (Bacillus anthracis) (Watson et al., 2004)

caF1-lcrV F1-V protein /Plague (Yersinia pestis) (Arlen et al., 2007)

ospA-histag lipoprotein A/Lyme disease (Borrelia burgdorferi) (Glenz et al., 2006)

lecA lectin A (Entamoeba histolytica) (Chebola and Daniell, 2007)

p24 p24 antigen (Human Immunodeficiency Virus) (McCabe et al., 2008)

L1 L1 protein (Human papillomavirus) (Lenzi et al., 2008)

Introduction

18

3.2.5 Plastid transformation in different species

Although plastid transformation in higher plants was established more

than 20 years ago, stable transformants could only be obtained in relatively few

species (Maliga, 2004). In order to obtain transplastomic crops, some critical points

must be considered. One of them is the design of specific vectors for each species

(Bock and Khan, 2004). Another critical point is the in vitro culture of crops,

allowing a high regeneration capacity so that fertile plants can be recovered

(Maliga, 2002; Koop et al., 2007).

Currently, stable plastid transformation was successfully achieved for solanaceous

crops using similar tissue culture conditions as for tobacco, potato, eggplant,

tomato and petunia (Svab et al., 1990; Sidorov et al., 1999; Ruf et al., 2001; Zubkot et

al., 2004; Singh et al., 2010; Valkov et al., 2010). Plastid transformation was also

described in other species as soybean/Glycine max (Dufourmantel et al., 2004);

carrot/Daucus carota (Kumar et al., 2004a); cotton/Gossypium hirsutum (Kumar et

al., 2004b); wheat/ Triticum aestivum (Cui et al., 2011); lettuce/Lactuca sativa

(Lelivelt et al., 2005; Kanamoto et al., 2006); rice/Oryza sativa (Lee et al., 2006); sugar

beet/Beta vulgaris (De Marchis et al., 2008) and poplar/Populus alba (Okumura et

al., 2006). Finally there are some reports in the Brassicaceae family: Lesquerella

fendleri (Skarjinskaia et al., 2003); rapeseed/Brassica napus (Hou et al., 2003; Cheng

et al., 2010), cabbage/(Liu et al., 2007)and cauliflower/Brassica oleracea (Nugent et

al., 2005a) and Arabidopsis thaliana (Sikdar et al., 1998). In Arabidopsis thaliana there

is just one report which describes unfertile plants. However, stable transformation

of chloroplast in Arabidopsis thaliana could not be reproduced in the last ten years

(Sikdar et al., 1998).

3.3 Arabidopsis thaliana as model organism

Arabidopsis thaliana is a member of the Brassicaceae family which also

includes some cultivated species such as cauliflower, radish and mustard.

Although Arabidopsis thaliana is not an agronomically important plant, several

Introduction

19

advantages made Arabidopsis thaliana one of the most important model organisms

in plant molecular biology (Bressan et al., 2001; Koornneef and Meinke, 2010).

First, Arabidopsis thaliana has one of the smallest genomes in the plant kingdom

with approximately 135 megabases. In addition, the nuclear, plastid and

mitochondrial genomes have been sequenced (The Arabidopsis Genome Initiative

2000) (Ausubel, 2000; Koornneef and Meinke, 2010).

The nuclear genome of Arabidopsis thaliana consists of 5 chromosomes identified by

Friedrich Laibach in 1907. Leibach is the founder of experimental research in

Arabidopsis thaliana and in 1943, he proposed Arabidopsis thaliana as a model

organism (Meyerowitz and Pruitt, 1985; Meyerowitz, 2001; Somerville and

Koornneef, 2002). It can be easily cultivated and develops rapidly, taking about 6

weeks from seed germination to the production of a new crop of seeds. Moreover,

Arabidopsis thaliana is a self-fertilizing diploid plant and can produce up to 50,000

seeds per plant.

The most common nuclear transformation method in Arabidopsis thaliana is the

“dipping method”. Here, the whole flowering plant is dipped into a solution

containing Agrobacterium tumefaciens with a T-DNA plasmid. The T-DNA plasmid

which contains the gene of interest is then introduced into cells by the

Agrobacterium tumefaciens method. Once transformed cells develop into gametes,

and the transgenic plants can be subsequently selected from the treated seeds

(Chang et al., 1994).

Mutations in Arabidopsis thaliana can be generated using x-ray irradiation, chemical

mutagens, insertional mutagenesis via Agrobacterium thumefaciens or transposons

(Meinke et al., 1998). Rapid isolation of mutants together with the simple

identification of the corresponding genes offers another advantage of Arabidopsis

thaliana.

Due to these properties, Arabidopsis thaliana has become one of the most important

model organism for the understanding of plant development, cell biology,

biochemistry and physiology (Meyerowitz, 2001; Koornneef and Meinke, 2010).

Introduction

20

3.3.1 Tissue culture in Arabidopsis thaliana

In most plants, tissue culture is critical for successful plastid transformation.

In vitro regeneration of plants requires a suitable medium containing mineral salts,

an energy source as sucrose, vitamins and phytohormones like cytokinins and

auxins (Murashige and Skoog, 1962). Because plants possess totipotency in most of

their cells, almost each individual somatic cell can regenerate a whole plant under

controlled conditions (Vasil and Vasil, 1972).

In vitro regeneration of plants can be achieved through two pathways, either by

somatic embryogenesis or by organogenesis. For somatic embryogenesis, plants

can be regenerated directly from explants or indirectly after a callus phase (group

of undifferentiated cells) (Figure 3.6).

In Arabidopsis thaliana somatic embryogenesis could be achieved from seed-

derived calli (Huang and Yeoman, 1984) and from immature zygotic embryo-

derived calli (Luo and Koop, 1997). Somatic embryos are bipolar, meaning that

they contain both shoot and root apices. Starting from cotyledon derived callus,

somatic embryogenesis can be induced by the addition of the synthetic auxin 2-4-

dichlorophenoxyacetic acid (2-4-D) (Raghavan, 2004).

For in vitro organogenesis in Arabidopsis thaliana, a two-step procedure is required.

First, to induce callus formation, the explants are cultured on an auxin-rich

medium. Then, for the induction of shoots, they are transferred to a cytokinin-rich

medium (Valvekens et al., 1988). Regenerated shoots can be finally transferred to

hormone-free medium or to a root induction medium to produce a complete plant.

De novo organogenesis involves three phases:

1. Acquisition of competence (dedifferentiation)

2. Induction

3. Morphological differentiation.

Introduction

21

Regeneration efficiency depends not only on tissue culture media, but it also

depends on the explant source and plant ecotype. Successful regeneration of

Arabidopsis thaliana has been achieved using different explants such as leaves

(Feldmann and Marks, 1986; Schmidt and Willmitzer, 1988; Candela et al., 2001),

cotyledons (Patton and Meinke, 1988; Barghchi et al., 1994; Zhao et al., 2002),

hypocotyls (Zhao et al., 2002), seeds (Negrutiu et al., 1975; Huang and Yeoman,

1984) and roots (Valvekens et al., 1988; Atta et al., 2009).

With regard to ecotypes, the highest regeneration efficiencies were reported in

ecotypes C24, Wassilewskija (WS) and Landsberg erecta (Ler) when compared

with other ecotypes as Columbia or RLD (Barghchi et al., 1994; Luo and Koop,

1997; Candela et al., 2001).

3.3.2 Protoplast culture in Arabidopsis thaliana

For protoplast isolation, the cell wall must be removed with fungal

hydrolytic enzymes such as cellulase and macerozyme. In Arabidopsis thaliana,

protoplasts have been isolated from several sources like leaves, cotyledons, roots,

cell suspension and callus. Once the tissue has been incubated with suitable

hydrolytic enzymes, protoplast isolation requires subsequent filtration, floatation

and sedimentations steps.

Arabidopsis thaliana protoplasts have been used for the understanding of cellular

processes including cell wall synthesis, cell division, photosynthesis activity,

calcium signalling, stress and responses to plant hormones (Yoo et al., 2007).

Figure 3.6 In vitro regeneration of Arabidopsis thaliana

Introduction

22

Nowadays, protoplast isolation and transient gene expression has become a

standard tool in plant physiology. Long standing challenges in this research field

thus far are high regeneration efficiencies of complete fertile plants from

protoplasts (Dovzhenko et al., 2003).

As for tissue culture, regeneration efficiency depends highly on the Arabidopsis

thaliana ecotypes, for instance, ecotype C24 was successfully regenerated with the

highest platting efficiencies of up to 30 % when compared with other ecotypes as

Wassilewskija with 10 % and Columbia with 5 % (Dovzhenko et al., 2003).

3.4 Rapid-Cycling Brassica rapa, a new model organism

Brassicaceae is a diverse group of crop plants economically important in

agriculture and horticulture. Brassicaceae are related to Arabidopsis thaliana and

includes vegetables as broccoli, cabbage, cauliflower, turnip and mustard

condiments, from which several uses include, besides human consumption,

animal feed and the production of industrial oil (Musgrave, 2000).

Brassicaceae include three diploid species, Brassica rapa or campestris (AA), Brassica

nigra (BB) and Brassica olaracea (CC) and three naturally occurring allotetraploid

species, Brassica juncea (AABB), Brassica napus (AACC) and Brassica carinata

(BBCC). The genetic relationship between the Brassica spp. is described by the so-

called "U´s triangle" proposed by Woo Jang-choon in 1935 (Hong et al., 2008)

(Figure 3.7).

Although Brassicas are a quite diverse and economically important group of

plants, there is relatively little knowledge on their genetics and molecular

physiology. This is due probably to its long reproductive cycles that last from 6

months up to a year for most of the horticultural types (Williams and Hill, 1986).

Introduction

23

Figure 3.7 The genetic relationship between Brassica species of the "Triangle of U". Diploid species are indicated as AA (Brassica rapa), BB (Brassica nigra), CC (Brassica olaraceae). Chromosomes from each of the genomes A, B and C are represented by different colours. “n” represent the number of chromosomes in the haploid genomes. Figure from http://canseq.ca/

To solve this long reproductive cycle problem, Williams et al., worked with a seed

bank of approximately 2000 accessions of Brassicas. They re-discovered Brassica

seeds from a mountainous region of Nepal 20 years ago. Williams et al., bred this

weedy Brassica until they were able to reduce its life cycle to 5 weeks under

standard conditions (www.fastplants.org). Nowadays, Rapid-Cycling Brassica rapa

(RCBr) known as Fast Plants® or Wisconsin plants are used in research and

teaching (Musgrave, 2000).

Characteristics of RCBr are:

- Short time from planting to flowering (about 2 weeks)

- Rapid seed maturation with no seed dormancy (35-45 days)

- Small plant size (up to 20 cm)

RCBr is naturally self-incompatible for pollination and they represent a genetically

diverse group with many different strains (Briggs and Goldman, 2006). All these

characteristics made RCBr a very attractive model for teaching and also for

learning genetics, ecology, botany and physiology (Wendell and Pickard, 2007). To

date over 150 different lines have been described (www.fastplants.org).

Introduction

24

4 Research aims

The main goal of this work was the establishment of protocols towards the

plastid transformation in Arabidopsis thaliana and the Rapid-Cycling Brassica rapa.

To achieve this goal, we focused on the generation of new vectors containing

species specific sequences for the integration of transgenes into the chloroplast

genome. Additionally, we concentrated on the standardization of tissue culture to

obtain high regeneration efficiencies of fertile plants.

Plastid transformation is a well-established method in Nicotiana tabacum, for this

reason tobacco plants were used as a positive control in the transformation

experiments.

Material and Methods

25

5 Materials and Methods

5.1 Chemicals and enzymes

2,4-dichlorophenoxy acetic acid (2,4-D) Sigma-Aldrich, Taufkirchen, Germany 3-Indole acetic acid (IAA)* Sigma-Aldrich, Taufkirchen, Germany Acetone Merck, Darmstadt, Germany Agarose (Seakem® LE agarose) Cambrex, Rockland ME, USA Alginic acid Sigma-Aldrich, Taufkirchen, Germany Ammonium succinate Sigma-Aldrich, Taufkirchen, Germany Ammonium sulphate* Duchefa, Haarlem, Netherlands Ampicillin, sodium salt AppliChem, Darmstadt, Germany B5 salts Duchefa, Haarlem, Netherlands Bacto Agar Difco, Detroit Michigan, USA Benzylaminopurine (BAP)* Sigma-Aldrich, Taufkirchen, Germany Boric acid USB, Cleveland, USA Bromophenol blue Merck, Darmstadt, Germany β-Mercaptoethanol Roth, Karlsruhe, Germany Calf intestine phosphatase Fermentas, St Leon-Rot, Germany Calcium chloride dehydrate* Merck, Darmstadt, Germany Calcium pantothenate Merck, Darmstadt, Germany Casein hydrolysate* ICN Biochemicals, USA Cellullase R10 Duchefa, Haarlem, Netherlands Cetyl trimethyl ammonium bromide (CTAB) Serva, Heidelberg, Germany Chloroform Roth, Karlsruhe, Germany D(+)-Biotin (vitamin B8)* Duchefa, Haarlem, Netherlands D-Glucose* AppliChem, Darmstadt, Germany Diaminoethanetetra acetic acid (EDTA) Serva, Heidelberg, Germany Dicamba* Sigma-Aldrich, Taufkirchen, Germany Dimanin C Bayer Vital, Leverkusen, Germany D-Mannose* Sigma-Aldrich, Taufkirchen, Germany DNA Dream Taq polymerase Fermentas, St Leon-Rot, Germany DNA Phusion polymerase Finnzymes, Espoo (Finnland) Ethidium bromide Sigma-Aldrich, Taufkirchen, Germany Folic acid* Duchefa, Haarlem, Netherlands Gelrite * Duchefa, Haarlem, Netherlands Glutamic acid Fluka, Buchs, Switzerland Glycerol Roth, Karlsruhe, Germany Glycine* Merck, Darmstadt, Germany Gold (0,6 μm) Biorad, Munchen. Germany Hydrochloric acid Sigma-Aldrich, Taufkirchen, Germany Indole butyric acid (IBA)* Merck, Darmstadt, Germany Isopropanol Duchefa, Haarlem, Netherlands Kanamycin Duchefa, Haarlem, Netherlands Kinetin Sigma-Aldrich, Taufkirchen, Germany L-arginin Sigma-Aldrich, Taufkirchen, Germany L-asparagin Sigma-Aldrich, Taufkirchen, Germany L-aspartic acid Sigma-Aldrich, Taufkirchen, Germany L-Glutamine Sigma-Aldrich, Taufkirchen, Germany L-proline* Duchefa, Haarlem, Netherlands Macerocyme R10 Duchefa, Haarlem, Netherlands Magnesium chloride hexahydrate AppliChem, Darmstadt, Germany


26

Magnesium sulphate heptahydrate Roth, Karlsruhe, Germany Magnesium sulphate monohydrate Merck, Darmstadt, Germany Manganese (II) chloride, tetrahydrate Roth, Karlsruhe, Germany Manganese sulphate monohydrate Merck, Darmstadt, Germany Mannitol Duchefa, Haarlem, Netherlands MES (2-(N-Morpholino)ethanol acid) Sigma-Aldrich, Taufkirchen, Germany Methanol (HPLC grade) Merck, Darmstadt, Germany MS salts Duchefa, Haarlem, Netherlands Myo-inositol * Serva, Heidelberg, Germany Naphthalene acetic acid (NAA)* Sigma-Aldrich, Taufkirchen, Germany Nicotinic acid Sigma-Aldrich, Taufkirchen, Germany Oligonucleotides Metabion, Martinsried, Germany Phyto agar* Duchefa, Haarlem, Netherlands Potassium acetate Merck, Darmstadt, Germany Potassium dihydrogen phosphate Merck, Darmstadt, Germany Potassium hydroxide Merck, Darmstadt, Germany Potassium iodide * Merck, Darmstadt, Germany Potassium nitrate Merck, Darmstadt, Germany Pyridoxine hydrochloride (vitamin B6)* Duchefa, Haarlem, Netherlands Riboflavin (vitamin B2)* Duchefa, Haarlem, Netherlands Rubidium chloride AppliChem, Darmstadt, Germany Silver nitrate* Roth, Karlsruhe, Germany Sodium acetate Roth, Karlsruhe, Germany Sodium chloride Sigma-Aldrich, Taufkirchen, Germany Sodium citrate Sigma-Aldrich, Taufkirchen, Germany Sodium dodecyl sulfate (SDS) Roth, Karlsruhe, Germany Sodium EDTA Roth, Karlsruhe, Germany Sodium hydroxide Merck, Darmstadt, Germany Sodium hypochlorite AppliChem, Darmstadt, Germany Spectinomycin Duchefa, Haarlem, Netherlands Spermidine Duchefa, Haarlem, Netherlands Sucrose* Duchefa, Haarlem, Netherlands Thiamine hydrochloride (vitamin B1)* Merck, Darmstadt, Germany Thidiazuron (1-Phenyl-3-(1,2,3-Thidizol) Sigma-Aldrich, Taufkirchen, Germany Tris(hydroxymethyl)-aminomethan (Tris) Roth, Karlsruhe, Germany Yeast extract Duchefa, Haarlem, Netherlands

* For plant in vitro-culture media.

All the DNA markers, restriction and nucleic acids modifying enzymes were

provided by MBI Fermentas (St. Leon-Rot, Germany), New England Biolabs

(Frankfurt Main, Germany) and Promega (Mannheim, Germany). Highly pure

deionized distilled water was obtained using an “Ultra Clear” SG equipment

(Barsbüttel, Germany).


27

5.2 Kits

NucleoSpin Plant Kit Macherey-Nagel GmbH & Co., Düren, Germany QIAquick Gel Extract Kit Qiagen, Hilden, Germany Plasmid Maxi Kit Qiagen, Hilden, Germany QIAquick Gel Extraction Kit Qiagen Qiagen, Hilden, Germany QIAquick PCR Purification Kit Qiagen, Hilden, Germany DIG High Prime DNA Labeling Starter Kit II Roche Diagnostics GmbH, Mannheim, Germany

5.3 Disposable material

Centrifugation tubes polyprop. (15, 50 ml) BD Falcon, Heidelberg, Germany Filter paper Whatman 3MM Whatman GmbH, Daasel, Germany Microcentrifugation tubes (1, 2 ml) Eppendorf AG, Hamburg, Germany Nescofilm Roth, Karlsruhe, Germany N+-Nylonmembran Amersham Biosciences, UK PCR tubes 0.5 and 0.2 ml DNase/RNase-free Peqlab GMBH, Erlangen, Germany Petri dishes for bacteriology, steril (9 cm) Renner, Dannstadt, Germany Petri dishes for tissue culture, steril (6 cm) Greiner, Solingen, Germany Petri dishes for tissue culture, steril (9 cm) Greiner, Solingen, Germany Scalpels, sterile and disposable Aesculap, Tuttlingen, Germany Steril filter PES, 0.22µm Serva, Heidelberg, Germany Steril filter PES, 0.22µm, 250 ml Serva, Heidelberg, Germany Syringes BD Plastipak, 1, 5 and 10 ml BD Biosciences Heidelberg, Germany Polypropylene grids Scrynel PP2000 K.H.Büttner, Wasserburg, Germany

5.4 Equipment and instruments

Analytical balance AC 120 S Sartorius, Göttingen, Germany Analytical balance 2255 Sartorius Sartorius, Göttingen, Germany Autoclave Aesculap 420 Aesculap-Werke, Tuttilngen, Germany Camera Canon EOS 400D +18-55/3,5-5,6 KIT Canon, Tokio (Japan) Centrifuge Universal 30 RF Hettich, Tuttlingen Centrifuge, refrigerated, model 5415 Eppendorf, Hamburg, Germany Centrifuge, Z323K Hermle, Wehingen, Germany Electrophoresis power supply 2301 LKB Produkter, Bromma, Sweden Electrophoresis chamber Peqlab Biotechnologie GmbH, Germany Fluorescence microscope „Axio Imager Z1” Carl Zeiss Vision GmbH, Jena, Austria Fluorescence microscope, Leica DM 1000 Leica Microsystems, Wetzlar, Germany Fuorescence SteREO Lumar. V12 Carl Zeiss Vision GmbH, Jena, Austria Forceps BD887R Aesculap, Tuttlingen, Germany Gel documentation system MWG Biotech, Ebersberg, Germany Growth cabinet Rumed Nr; 1200 Rubarth Apparate, Hannover, Germany Haemocytometer Fuchs-Rosenthal Friedrichsdorf, Germany Osmometer O30 Gonotec GmbH, Berlin, Germany Particle gun PDS/1000/He Bio-Rad, München, Germany PCR Express Thermocycler Hybaid Hybaid, Ashford, UK pH meter WTW pH522 WTW, Weilheim, Germany Pipettes Pipetman 2,20,200, 1000 µl Gilson, Middleton, Wisconsin, USAei Ultraspec 3100 UV/Vis Spectrophotometer GE Healthcare, UK


28

Sterile bench UVF6.12S BDK BDK, Sonnenbühl-Genkingen, Germany UV-Crosslinker Amersham, Freiburg, Germany Vacuum Concentrator Bachofer, Reutlingen, Germany Vortex mixer Genie 2 Modell 6560 Scientific Ind., Bohemia, NY, USA Water bath Wagner Wagner Munz, München, Germany

5.5 Software and internet tools

Function Name Reference Sequence Blast 2 Sequences (NCBI) http://www.ncbi.nlm.nih.gov/BLAST/ Primer design Primer3 Input 0.4.0 http://frodo.wi.mit.edu/ Vector design Vector NTI Version 4.0.2 Invitrogen, Groningen, Netherlands Gel analysis OneDScan MWG Biotech, Ebersberg, Germany Pictures Adobe Photoshop 6.0 Adobe Systems, San Jose (USA) AxioVision LE, Release 4.4 Carl Zeiss, Munich, Germany Office Software MS Office 2003-2007 Microsoft, Redmond (USA)

5.6 Biological material

5.6.1 Plant material

Nicotiana tabacum cv. Petit Havana was used as a control of plastid

transformation. Arabidopsis thaliana and Rapid-Cycling Brassica rapa (Fast plants®)

were used for standardization of tissue culture and towards plastid

transformation experiments (Table 5.1).

Table 5.1 Plant material

Plant Description Abbreviation Provided

Arabidopsis Arabidopsis thaliana C24 C24 PD Dr. Jörg Meurera Brassica Fast plants®

Rapid-Cycling Brassica rapa C1 RCBr-C1 College of Agricultural & life sciencesb

Rapid-Cycling Brassica rapa B3 RCBr-B3 Assistent Scientist S. Woodyc

a Botanic Institute, Ludwig-Maximillians-University, Munich, Germany b Department of Plant Pathology , University of Wisconsin-Madison, USA c Department of Biochemistry, University of Wisconsin-Madison, USA.

5.6.2 Medium for culture of bacteria

Medium, required for culture of Escherichia coli:

LB(Luria-Bertani) broth medium

Bacto-tryptone 10g

Bacto-yeast extract 5 g

NaCl 10g

pH to 7.0 with 1 N KOH, H2O add 11. Sterilize by autoclaving for 20 minutes.


29

Escherichia coli, XL1-Blue (genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1

lac [F´proAB lacIqZΔM15 Tn10 (Tetr)]), was used for cloning experiments. The

bacteria were grown overnight on either in LB broth or in Petri dishes containing

solidified LB (1.2 % Bacto agar) at 37 ºC. Appropriate antibiotics at suitable

concentration were used as described previously (Sambrook et al., 2001).

5.7 Molecular methods

5.7.1 Isolation of genomic DNA of plants

Plant genomic DNA was extracted using the NucleoSpin Plant Kit

(Macherey-Nagel, Düren, Germany) as described in the user protocol, or with the

modified CTAB method (Murray and Thompson, 1980) using the following

extraction buffer:

2x CTAB-Extraction buffer

Tris-HCl (pH 8) 200 mM CTAB 2 % (w/v) EDTA 20 mM NaCl 1400 mM PVP 1 % (w/v) β-Mercaptoethanol 280 mM (before each use)

Plant tissue (100-200 mg leaf or calli) was ground in liquid nitrogen to a fine

powder by using a mortar and a pestle. Plant material was homogenized in one

volume of CTAB (2X) extraction buffer and incubated for one hour at 60ºC. For

extraction, one volume of chloroform was added and tubes were incubated with

orbital agitation for 30 min. All tubes were then centrifuged for 5 min, at 10 000 g.

The upper layer was transferred to a sterile 2 ml Eppendorf tube and 5 µl RNAse

A [10 µg/ml] were added. After incubation for 30 min at 42 ºC, chloroform

extraction was repeated. The upper layer was transferred again to a new

Eppendorf tube and 0.7 volume of cold 2-Isopropanol (100 %) was added. The

DNA samples were incubated at -20 ºC for 30 min, and then centrifuged 10 min at

21 000 g. The supernatant was discarded; the DNA pellet was washed with cold

70 % EtOH solution, dried and dissolved in 50 µl of 10 mM Tris-HCl, pH 8.


30

5.7.2 Isolation of plasmid DNA

Plasmid DNA isolation was performed either on small or big-scale. The big-

scale set up was required for transformation experiments with the particle gun

method. Small-scale plasmid DNA preparations were used for cloning and sub-

cloning experiments. DNA mini and maxi preparations were performed using the

QIAprep Spin Miniprep Kit and the Plasmid Maxi Kit (Qiagen, Hilden, Germany),

respectively, according to the manufacturer´s protocol.

As an alternative, the “TENS” method was used. The TENS method is a

“rapid alkaline extraction” method (Birnboim and Doly, 1979) that has been

modified according to (Eibl, 1999). First, 3 ml of LB medium with appropriate

antibiotic concentration was inoculated with a single E. coli (XL1-Blue) colony and

incubated on a shaker at 37 ºC overnight. The next day, 2 ml of this culture were

centrifuged at 15 000 rpm for 2 min and the pellet was resuspended in 300 µl of

TENS buffer (50 mM Tris-HCl pH 7.5, 0.5 % SDS, 50 mM EDTA, 10 mM NaCl) by

vortexing. Then, 150 µl of KoAc 3M (pH 5.2) were added and the samples were

mixed by vortexing for 2-5 seconds. Cell debris and chromosomal DNA were

pelleted by centrifugation at 15 000 rpm for 4 min. The supernatant was

transferred into a new 1.5ml Eppendorf tube and 900 µl EtOH were added to

precipitate the plasmid DNA, supported by incubating at –20 °C for 5 min. After

centrifugation (15 000 rpm, 10 min), and washing with 70 % EtOH, the air-dried

pellet was finally dissolved in 50 µl of water or in 10 mM Tris-HCl, pH 8.

5.7.3 Gel electrophoresis

Agarose gel electrophoresis was used to separate DNA fragments according

to their sizes. Agarose gels were prepared at a final concentration of 1 % in a

buffer solution, either TAE 1X (40 mM Tris, 0.11 % acetic acid (v/v), 1 mM

Na2EDTA) or TBE 1X (90 mM Tris, 90 mM Boric acid, 2 mM EDTA) buffer. To

dissolve the agarose, an agarose solution was boiled, and once this solution was

cooled down, 2.5 µl of 10 mg/ml ethidium bromide (EtBr) stock solution was

added to 50 ml. The melted gel was poured into a gel chamber with a comb. The


31

comb was removed after solidification and running buffer was added to the

chamber. Before loading, samples were mixed with 6X loading buffer (0.25 %

bromophenol blue, 0.25 % xylene cyanol, 30 % glycerin, 0.12 M Na2EDTA, pH 8.0).

Electrophoresis was performed at 60-120 V. A 1kb DNA ladder or a λ/HindIII

marker was included. DNA was indirectly visualized by UV light (254-366 nm).

Ethidium bromide intercalates between the DNA base pairs, and it fluoresces

under UV radiation (Sambrook et al., 2001). Results were scanned using a gel

documentary system (MWG, Ebersberg, Germany).

5.7.4 DNA purification and concentration

DNA purification was necessary after enzyme treatment of PCR

amplification. After enzymatic digestions, DNA was loaded in a gel and separated

according to size (see 5.7.3). Fragments of interest were excised and gel DNA

extraction was performed using the QIAquick Gel Extract Kit (Qiagen, Hilden,

Germany) according to manufacturers´ instructions. After a PCR or treatment of

DNA with any modifying enzyme, the PCR Extract Kit (Qiagen, Hilden, Germany)

was used as described in the user´s protocol.

DNA was subsequently concentrated by EtOH precipitation (Sambrook et al.,

2001). In this case, 2.5 volumes of 100 % of EtOH were added together with

0.1 volume of 3M sodium acetate (pH 4.8) followed by mixing. This solution was

incubated for 1 to 2 hours at -20 ºC and then centrifuged (12 000 rpm for 10 min).

The pellet was once washed using 70 % EtOH and then air-dried and dissolved in

10mM Tris-HCl pH 8.0.

5.7.5 DNA quantification

For DNA quantification, agarose electrophoresis densitometry was chosen to

estimate DNA integrity. Samples were loaded in a TBE 1X agarose gel (1 %)

according to section 5.7.3. As standard, a λ/HindIII marker was used. 6 µl of this

marker contains bands with the following known DNA amounts: 22, 24, 69, 100


32

and 240 ng. Pictures in “TIF” format from the gel were taken using a gel

documentation system and analyzed using the OneDScan (MWG, Ebersberg,

Germany) program.

5.7.6 DNA restriction and modification

For cloning experiments, analysis of vectors and transplastomic plants,

restriction enzymes were used. The reaction conditions were used as suggested by

the manufacturers´ following a standard protocol: 1 U of the enzyme for

1 µg DNA was supplemented with the appropriate buffer and incubated for 2 h.

Reaction products were analyzed by gel electrophoresis (see 5.7.3).

Blunt ends can be generated by using the Klenow fragment (the large fragment of

DNA polymerase I from E. coli) (Fermentas, St. Leon-Rot, Germany). The Klenow

polymerase I exhibits 5’3’ polymerase activity and 3’5’ exonuclease

(proofreading) activity but lack a 5’3’ exonuclease activity. After DNA digestions

with endonucleases were heat inactivated according to the manufacturers´

instructions. To fille of the 5’-overhangs, 1 µl of a dNTP mix (2.5 mM each) and

2 U of Klenow fragment were added to the sample and incubated at 25 ºC for

15 min. The enzyme was inactivated by adding 10 mM EDTA followed by heating

at 75 ºC for 20 min.

To minimize self-ligation, the termini of the blunted vectors were

dephosphorylated with the enzyme SAP (Shrimp Alkaline Phosphatase) enzyme

from Fermentas (St. Leon-Rot, Germany). Buffer conditions and temperature were

used according to the manufacturers´ advice.

5.7.7 DNA ligation

Ligation of two double-stranded DNA fragments with cohesive or blunt

termini was achieved using T4-DNA ligase from Fermentas (St. Leon-Rot,

Germany). In most cases, 20 ng of vector were used with a 3:1 or 5:1 vector/insert

ratio. The following formula was used to calculate this ratio: [(ng vector) x (kb size


33

of insert)]/[(kb size of vector)] x [molar ratio of (insert/vector)] = ng insert. Buffer

and incubation temperature were used according to manufacturers´ instructions.

5.7.8 Preparation of competent cells

Heat-shock Rb+ competent cells of E. coli XL-1Blue were prepared following a

Hanahan D., (1983) modified method (Hanahan, 1983).

Solutions:

RF1 RF2

100 mM RbCl 10 mM RbCl 50 mM MnCl2·6H2O 75 mM CaCl2 30 mM KoAc 15 % Glycerol (v/v) 10 mM CaCl2 pH 6.8 with NaOH 15 % Glycerol (v/v) 10 mM MOPS (pH 6.5 with KOH) pH 5.8 with CH3COOH

A 5 ml LB starter culture was inoculated with 50 µl of a frozen stock of XL1-Blue

cells. The culture was incubated overnight at 37 ºC with constant shaking at

150 rpm. On the following day, a small aliquot equivalent to an OD600= 0.1 was

poured into 100 ml of fresh LB medium. Bacteria were grown until an OD600= 0.45-

0.6 was reached. Then, cells were incubated 2 h on ice and centrifuged for 10 min

at 7000 rpm. The pellet was dissolved in 20 ml of RF1 solution and incubated again

for 2 h on ice. Next, cells were centrifuged for 10 min at 7 000 rpm and the pellet

was dissolved in 4 ml of RF2 solution. Finally, aliquots of 50 or 100 µl were frozen

in liquid nitrogen and stored at -70 ºC.

5.7.9 Heat shock transformation of Escherichia coli

An aliquot of 50 µl competent cells of E. coli XL-1Blue was placed on ice for

20 min. Afterwards, a suitable DNA concentration was added (< 10 µl) to the cells;

the mixture was swirled and incubated on ice for 15 min. The mixture was heat-

shocked without agitation at 42 ºC for 60-90 seconds and placed on ice for 2-5 min.

Then, 800 µl of LB medium were added to the tube and incubated at 37 ºC, with


34

constant agitation at 200 rpm for 1 hour. Finally, 100 to 200 µl of the culture were

spread on an LB plate with proper antibiotics and the plate was incubated at 37 ºC

ON.

5.7.10 PCR and sequencing

PCR was used to generation of plastid transformation vectors and for the

characterization of transplastomic plants. To amplify DNA with high fidelity, the

Phusion polymerase (Finnzymes, Espoo, Finnland) was used while the Dream Taq

DNA polymerase from Fermentas (St. Leon-Rot, Germany) was used for standard

PCR amplification according to the instruction manual. For the reaction mixture, a

25 mM dNTP mix was prepared from 100 mM dNTP´s set of Fermentas (St. Leon-

Rot, Germany). Oligo primers were designed with the help of Primer 3 Input 0.4.0

software and synthesized by Metabion company (Martinsried, Germany). PCR

amplifications were carried out in a Hybaid thermocycler (Heidelberg, Germany)

with reaction settings and programming parameters as described in Tables 5.2 and

5.3. After amplification, 5 µl of each sample were characterized by gel

electrophoresis.

Table 5.2 The reaction settings of PCR amplification

Components \ applications DNA amplification with high fidelity

Identification of transplastomic plants

10 pmol/µl Primer (P1) 10 pmol/µl Primer (P2) 10 mM dNTP mixture 10 X or 5 X reaction buffer Plant total DNA (50 ng~200 ng/µl) Taq DNA polymerase (5 U/µl) Phusion DNA polymerase (3 U/µl) Distilled H2O Total volume

2.5 µl 2.5 µl 1 µl 10 µl 2 µl --- 0.5 µl 31.5 µl

50 µl

1 µl 1 µl 1 µl 2.5 µl 2 µl 0.2 µl --- 19.3 µl

25 µl

Table 5.3 The program settings of PCR amplification

Cycles Applications DNA amplification with high fidelity

Identification of transplastomic plants

1 cycle 35 cycles 1 cycle

Pre-denaturing Denaturing Annealing Polymerization Extension

98 ºC 4 m 98 ºC 30 s 57 ºC 30 s 72 ºC 30 s 72 ºC 10 m

94 ºC 4 m 94 ºC 30 s 55-60 ºC 30 s 72 ºC 30 s 72 ºC 10 s


35

DNA sequences were analysed, using the in-house sequencing facility

(Martinsried, Germany).

5.7.11 Southern blot hybridization

Southern blots were carried out with the non-radioactive DIG method (DIG

High Prime DNA Labeling and Detection Starter Kit II) from Roche (Mannheim,

Germany), or with the standard radioactive method (Sambrook et al., 2001). In

either case 1-2 µg of probe were labeled according to the manufacturers´

instructions or to the Sambroock et al., (2001) instructions.

DNA was isolated from leaves of transplastomic plants growing in the culture

chamber. About 2 µg of total DNA of each sample were completely digested with

enzyme Kpn21 at 55 ºC or NsiI at 37 ºC for 3-4 hours.

A 1 % agarose gel was prepared in TPE buffer (360 mM Tris, 300 mM

Na2HPO4 H2O, 70 mM EDTA) containing 0.5 mg/ml EtBr. The DNA digestion

samples were loaded in a mixture with 6x DNA loading buffer in the gel slot and

ran at 30-40 V overnight for optimal separation. The gel was documented and

washed with distilled water. The gel was depurinated by submersion in 100 ml of

0.5 M HCl, in constant shaking at room temperature until the bromophenol blue

marker changed from blue to yellow (10-15 min). The gel was then rinsed with

double distilled water and denatured by incubating it twice with gentle shaking in

a denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 15 min at room temperature.

Gels were immerged in a neutralization solution (0.5 M Tris-HCl, pH 7.5;

1.5 M NaCl) two times, 15 min each, at room temperature with gentle shaking.

Afterwards the gel was equilibrated for at least 10 min in 20 X SSC buffer

(3 M NaCl, 0.3 M Na Citrate (NaH2C6H5O7), pH 7.0). For the transference of DNA

from the gel to the membrane, a DNA blot unit (Figure 5.1), was prepared in the

following way: about 8 pieces of Whatman 3 MM paper were soaked in 20 X SSC

and used to form a “bridge” with one part of this paper in a shallow reservoir of

20 X SSC. The gel was placed on the top of the soaked sheets of Whatman 3 MM


36

paper. A sterile pipette was rolled over the gel to remove air bubbles. A piece of

Hybond-N+ nylon membrane (Amersham, Braunschweig, Germany) was cut to

the size of the gel and incubated for 5 minutes in 20 X SSC. The membrane was

then placed above the gel and the pipette was used again to eliminate air bubbles

as mentioned above. The blot assembly was completed by addition of a dry sheet

of Whatman 3 MM paper, and a stack of paper towels, and finally a piece of 200 –

500 g weight on top. After 12-18 hours of overnight transfer, the Hybond-N+

nylon membrane was exposed to UV using the UV crossinglinker (Stratagene,

Heidelberg, Germany) for DNA fixation.

The membrane was briefly rinsed in double distilled water. To prepare the

hybridization buffer, 64 ml of sterile double distilled water were added to the Dig

Easy Hyb granules (bottle 7) to dissolve them by stirring for 5 min at 37 ºC. Then,

30 ml of DIG-Easy Hyb buffer prewarmed to 45 ºC were added to the membrane

and incubated at the same temperature for 1h in a rolling bottle incubator. The

hybridization solution was prepared as follows: 20 µl of the labeled probe were

incubated for 5 minutes in boiling water for denature and then the probe was

chilled quickly on ice. Immediately, the labeled probe was added to a tube,

containing 10 ml of prewarmed DIG-Easy Hyb buffer and mixed gentle by

inversion. The DIG-Easy Hyp buffer was removed from the bottle, and 10 ml of

Figure 5.1 Southern blot tray (Image from www.gbiosciences.com)


37

hybridization solution was added into the bottle containing the membrane.

Hybridization was done at 45 ºC overnight, in a rolling bottle incubator.

Detection:

Solutions:

Low Stringency buffer: 500 ml (2 X SSC + 0.1 % SDS): 50 ml 20 X SSC + 5 ml 10 % SDS High Stringency buffer: 500 ml: (0.5 X SSC + 0.1 % SDS): 12.5 ml 20 X SSC + 5 ml 10 % SDS Maleic acid buffer: 0.1 M Maleic acid, 0.15 M NaCl, pH 7.5 with NaOH Blocking solution: 12 ml 10 X Blocking solution + 108 ml Malein acid buffer. Washing buffer: 0.1 M Maleic acid, 0.15 M NaCl, pH 7.5 + 0.3 % Tween 20 Antibody solution: Centrifuge Anti-Digoxigenin-AP for 5 min at 10 000 rpm in the

original vial prior to each use, and pipet 2 µl in 20 ml of Blocking solution 1 X (dilution 1:10 000 – prepare fresh 2h, 4 ºC-).

Detection buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20 ºC)

Detection was realized as follows


38

5.8 Plant tissue culture

Stock solutions for plant tissue culture were prepared with inorganic mineral

elements (Table 5.4) and vitamins (Table 5.5).

Table 5.4 Composition of basal plant tissue culture media*

Components in g/l MS

(Murashige

and Skoog,

1962)

MS mod

(Dovzhenko

et al., 1998)

B5

(Gamborg

et al.,

1968)

B5 (G&E)

(Gamborg and

Eveleigh.,

1967)

RS

(Dovzhenko,

2001)

G & D

(Gresshoff and

Doy, 1972)

M

AC

RO

ELEM

EN

TS

10

X

KNO3 19 10.12 25 30 100

CaCl2·2H2O 4.4 4.4 1.5 0.15

MgSO4·7H2O 3.7 3.7 2.5 12.33 1.7

KH2PO4 1.7 1.7

NaH2PO4·H2O 1.5

NH4NO3 16.5 100

(NH4)2SO4 1.34

NH4PO4

Ca(NO3)2·H2O 0.708 23.6

KCl 6.5

KH2PO4 0.170 30

NaNO3 0.170

MIC

RO

ELEM

EN

TS

10

0 X

NaEDTA 4 4 4

KI 0.083 0.075 0.080

H3BO3 0.620 0.300 0.300 0.03

MnSO4·H2O 2.23 1 1 0.1

ZnSO4·7H2O 0.860 0.200 0.300 0.030

Na2MoO4·2H2O 0.025 0.025 0.025 0.0025

CoCl2·6H2O

CuSO4·5H2O

0.0025

0.0025 0.0025

0.0025

0.025

0.034 0.0025

0.0025

* All components were prepared with distilled water and stored as 100 ml aliquots at -20 ºC.

Table 5.5 Amino acids and Vitamins

Components in

g/l (100X)

MS

(Murashige and

Skoog, 1962)

B5

(Gamborg

et al.,

1968)

PC

(Glimelius

et al.,

1986)

NT

(Nagata and

Takebe,

1971)

Cocktail 20

(Mere-Villanueva

and Vazquez-

Alejandro, 2003)

Ara

(Márton and

Browse,

1991)

Glycine 0.200 2.3 0.100 myo - Inositol 10 10 20 10 14.5 10 Nicotinic acid 0.050 0.100 0.200 0.150 0.050 Pyridoxine HCl 0.050 0.100 0.200 0.150 0.050 Thiamine HCl 0.010 1 0.100 0.100 0.300 0.500 Biotin 0.002 0.100 0.005 L - asparagine 1 L - arginine 1 L - aspartic acid 0.750 Glutamine 0.60 Glutamic acid 0.750 Folic acid 0.100 Riboflavine 0.010 Urea 4.5 CaCl2·2H2O 20 Ca-Pantothenate 0.200

* All components were prepared with distilled water and stored as 100 ml aliquots at -20 ºC.


39

5.8.1 Shoot induction from seeds and cotyledons in

Arabidopsis thaliana

Seeds from Arabidopsis thaliana C-24 were surface sterilized using the Cl gas

sterilization method. About 30-40 mg of seeds were transferred into a 3 cm glass

Petri dish and four Petri dishes were placed with their lid open in a desiccator

under a fume hood. A beaker containing 100 ml of Calcium hypochlorite was

placed in the desiccator next to the seeds. Then, 3-5 ml of concentrated HCl were

added to the beaker to create chlorine gas. The desiccator jar was quickly sealed

and in this way, the seeds were exposed to the chlorine gas for about 4 h. Finally,

the Petri dishes containing seeds were transferred to a laminar flow hood and

ventilated with the lid open for 1 h.

The composition of culture media used for plant regeneration from Arabidopsis

thaliana is showed in Table 5.6. Induction and growth of calli from seeds was

performed in a climate chamber with diurnal cycle of 16 h light (60 μmol/m2s) at

25 °C followed by 8 h of darkness. Different PG callus induction media modified

from (Negrutiu et al., 1975) were used. To induce callus formation, PG1KN,

PG1KD, PG20KN and PG20KD media were used as well as ARMI media (Márton

and Browse, 1991). Upon incubating for four weeks, calli were transferred to the

regeneration media SRA2N (Zhao et al., 2002), SRAKN (Luo and Koop, 1997) and

ARMIIc (Márton and Browse, 1991). Finally, for seed production, regenerated

shoots were transferred to both root induction medium (RIM) (Luo, 1997) and

seedling culture Arabidopsis thaliana medium (SCA) (Dovzhenko et al., 2003).

The method of shoot regeneration from cotyledon explants has been modified

from Zhao et al., (Zhao et al., 2002). Surface sterilized seeds were transferred into

SCA medium and stored at 4 ºC for 1-3 days. Germination was performed at a

photoperiod of 16 h of light (130 μmol/m2s) at 22 °C followed by 8 h of darkness

at 20 °C. Cotyledons from 3, 6 and 8 days old seedlings were excised and

transferred to callus induction medium (CIM). Explants were incubated for 2 days

with 16 h light (100 μmol/m2s) at 22 ºC and 8 h dark. Cotyledons were transferred

into a regeneration medium (SRAKN or SRA2N) under the same photoperiod


40

conditions. Finally, shoots were transferred to either RIM or to SCA medium for

seed production.

Table 5.6 Germination and calli induction media in Arabidopsis thaliana*

*All media were adjusted to pH 5.8 and fill up to 1 l with distilled water, media were sterilized at 120 ºC for 20 min.

Table 5.7 Shoots and roots induction media in Arabidopsis thaliana*

* All media were adjusted to pH 5.8 and fill up to 1 l with distilled water; media were sterilized at 120 ºC for 20 min.

Components/l SCA PG1KN PG1KD PG20KN PG20KD CIM ARMI

B5 salts 3.1 g

MS salts 4.3 g 4.3 g

G&D Macro 10X 100 ml 100 ml 100 ml 100 ml

B5 (G&E) Micro 100X 10 ml 10 ml 10 ml 10 ml

B5 Vit 100X 10 ml

MS Vit 100X 10 ml

Ara Vit 100X 10 ml 10 ml 10 ml

Cocktail 20 100X 10 ml 10 ml

MgSO4·7H2O 1.14 g

2,4 D (1mg/ml) 1ml 1ml 150 µl

Kin (1mg/ml) 50 µl 50 µl 50 µl 50 µl

BA (1mg/ml) 600 µl

2ip (1mg/ml) 100 µl 300 µl

NAA (1mg/ml) 8 ml 8 ml 500 µl

IAA (1 mg/ml) 3 ml

Sucrose 20 g

Glucose 20 g 20 g 20 g 20 g 30 g

Agar 8 g 8 g 8 g 8 g 6 g 8 g

Gelrite 3 g

Components/l SRAKN SRA2N ARMIIc RIMI RIMII

MS salts 4.3 g 4.3 g 4.3 g 4.3 g 4.3 g

MS Vit 100X 10 ml 10 ml 10 ml 10 ml

Ara Vit 100X 10 ml

Kin (1mg/ml) 2 ml

2ip (1mg/ml) 900 µl 3 ml

NAA (1mg/ml) 50 µl 100 µl 100 µl 1 ml

IBA (1mg/ml) 1 ml 500 µl

Sucrose 30 g 30 g 30 g 30 g 15 g

Agar 6 g 6 g 8 g 8 g 8 g


41

5.8.2 Protoplast isolation, embedding and culture in


Protoplast culture was modified from Dovzhenko et al., (2002). About 30-40 mg

seeds were surface sterilized (see 5.8.1). Seeds were transferred to a 9 cm Petri dish

containing SCA medium and stored at 4 ºC for 1-3 days to break dormancy.

Germination was performed with 16 h of light (130-150 μmol/m2s) at 22 °C

followed by 8 h dark at 22 °C, during 6-7 days. The seedlings were incubated at

4 ºC in darkness during 1-3 days.

The media, employed for protoplast cultures are listed in Table 5.8. For pre-

plasmolysis, cotyledons were excised and placed in a Petri dish containing 5 ml of

MMC medium. After one hour incubation, cotyledons were cut into small pieces

with a scalpel, transferred to a new Petri dish and washed once with 1-2 ml of

MMC medium.

Subsequently, the MMC medium was removed and cotyledons were incubated in

5 ml of MMC supplemented with 50 mg/l cefotaxime, 0.5 % of cellulase R10 and

0.5 % of macerozyme R10 (10 % stock solutions of cellulase R10 and mecerozyme

R10 were prepared with 13.4 % of sucrose in sterile distilled water, and sterilized

by filtration) in the dark at 25 C for 12-16 h (Figure 5.2).

Afterwards, the incubation mixture was passed through a stainless steel sieve

(pore size, 100 m). The filtrate was adjusted to 10 ml with MMC and centrifuged

for 10 min at 50 g. The pellet was then resuspended in 10 ml of MSC medium, and

2 ml MMM medium were layered on top. After floatation (Figure 5.3), for 10 min

Figure 5.2 Cotyledons of Arabidopsis thaliana prepared for enzyme digestion. (a) Preincubation of cotyledons on a 6 cm Petri dish containing MMC medium. (b) Incubation of cotyledons on a Petri dish containing MMC medium with macerozyme R10 and cellulase R10.

(a) (b)


42

Debris cells

Intact protoplasts

at 70 g, protoplasts were collected from the interface and transferred to a new

centrifuge tube.

The volume was adjusted to 10 ml with MMM. From this suspension, 20 l were

transferred to a Haemocytometer (Fuchs-Rosenthal, Germany). Protoplasts on

5 big squares were counted and the average was calculated. To determine

protoplast density, the average was multiplied with the dilution factor (10 ml=

50,000). Thereafter, protoplast suspension was pelleted for 10 min at 50 g, the

supernatant was discarded and the protoplasts were resuspended in MMM and

mixed with the same volume of F-Alginat at a density of 1-1.5 X 105 pp/grids.

Protoplasts were immobilized in TAL (Thin Alginate Layers) as described by Luo

and Koop (1997), 625 µl of protoplast alginate mixture were transferred to the

surface of a Petri dish containing Ca-agar. A polypropylene grid of 10x10 meshes

(Büttner, Wasserburg, Germany) was inserted into the protoplast-alginate mixture

and incubated for 30 min.

After alginate solidification, grids with embedded protoplasts were placed upside

down into Petri dishes containing 10 ml of Arabidopsis thaliana culture medium

(PCA) for 30 min. Grids were subsequently transferred to new Petri dishes (6 cm)

with 4 ml PCA medium. After formation of protoplast derived colonies, grids

were placed on 9 cm Petri dishes containing SRAKN media. The regenerated

shoots were rooted on RIM medium.

Figure 5.3 Protoplast floatation. Isolation of intact protoplast after 10 min centrifugation at 70 g


43

Table 5.8 Media to isolation and embedding of Arabidopsis thaliana protoplasts

Medium Reference Purpose Basal medium Compositions (mg or ml/l)

MMC Luo, 1997 Protoplast floatation

--- 1.952 g MES 1.47 g CaCl2·2H2O

85 g mannitol to 600 mOsm MMM Dovzhenko et al.,

1998 Protoplast embedding

--- 1.952 g MES 1.020 g MgCl2· 6H20 1.250 g MgSO4·7H2O

85 g mannitol to 600 mOsm

MSC Luo, 1997 Protoplast floatation

--- 1.952 g MES 1.470 g CaCl2·2H2O

135 g sucrose to 600 mOsm F- Alginat Luo, 1997 Protoplast

embedding 1.370 g MES

2.500 g MgSO4·7H2O 2.040 g MgCl2·6H20

80 g mannitol to 600 mOsm 2.800 g alginate

Ca-Agar Luo, 1997 Protoplast embedding

2.940 g CaCl2·2H2O 10.00 g MES

85 g mannitol to 600 mOsm 10.00 g Agar

PCA Modified from Dovzhenko et al., 2003

Protoplast culture in Arabidopsis

B5 Salts (3.1g) B5 Vitamins 100 X (10 ml)

0.746 g MgSO4·7H2O 0.450 g CaCl2·2H2O 0.050 g L-glutamine 20.00 ml Coconut water 500 µl NAA (1mg/ml) 100 µl 2ip (1mg/ml)

85 g Glucose to 600 mOsm All media were adjusted to pH 5.8. F-Alginat and Ca-agar were autoclaved, and the other media were sterile filtrated.

5.8.3 Plant tissue culture in Rapid-Cycling Brassica rapa

Seeds from Rapid-Cycling Brassica rapa (RCBr) C1 and B3 were surface

sterilized with 70 % ethanol (v/v) for 1 min and then treated with 5 % (w/v)

Dimanin C and one drop of Tween-20 for 10 min. After this treatment, seeds were

washed three times with autoclaved distilled water for 10 min.

Media used for RCBr regeneration are described in Table 5.9. For each

regeneration experiment, approximately 10 seeds were germinated on solid MS

medium with 16 h of light (130 µmol/m2s) at 22 ºC followed by 8 h of darkness at

22 °C, or in RS medium with 16 h light (60 µmol/m2s) at 25 ºC followed by 8 h in

the dark. Calli were induced from various explants. Cotyledons, hypocotyls, and

roots were cut from 7 and 14 days old seedlings. For explant preparation, a

stereoscope Stemi SR (Zeiss, Göttingen, Germany), was used. Cotyledon and root

explants were prepared in a way that prevents presence of apical meristems


44

(Figure 5.4). Hypocotyls explants of approximately 2-4 mm size were cut at their

sagittal axis to expose the vascular tissues.

For regeneration experiments, around 20 segments were transferred to a Petri dish

containing MD1 or MDK media with 16 h of light (60 µmol/m2s) at 25 ºC followed

by 8 h of darkness. After 2-4 days, hypocotyl explants in MD1 medium were

transferred to BD1 medium keeping the same growth conditions for calli

induction. After 7 days on BD1 medium, explants were transferred to SIM

medium for two weeks for the induction of shoots. For the induction of shoots,

explants on MDK medium, were transferred to an MZI medium after two weeks

and kept there for additional three weeks. For roots formation, shoots were

transferred to RIM medium. Finally, after rooting, plants were transferred to RS

medium to allow seeds formation or to the greenhouse with photoperiod of 16 h

of light (60 µmol/m2s) at 25 ºC followed by 8 h of darkness.

Table 5.9 Rapid-Cycling Brassica rapa tissue culture media

Components/l MS RS MDN

(Teo et al.,

1997)

MD1 BD1 SIM

(Jonoubi et al., 2005)

MDK* MZI*

(Chen and Celio, 2000)

MS Salts 4.3 g 4.3 g 4.3 g 4.3 g 4.3 g B5 Salts 3.1 g 3.1 g

RS Macro 10 X 100 ml MS Micro 100 X 10 ml

MS Vitamins 100 X 10 ml 10 ml 10 ml 10 ml 10 ml B5 Vitamins100 X 10 ml 10 ml

Myo inositol 100 mg Thiamine- HCl 1.3 mg

KH2PO4 200 mg

2,4 D (1mg/ml) 1 ml 1 ml 500 µl BA (1mg/ml) 4.5 ml 4.5 ml Kinetin (1mg/ml) 200 µl IAA (1mg/ml) 500 µl Thidiazuron (1mg/ml) 300 µl Zeatin (1mg/ml) 2 ml NAA (1mg/ml) 400 µl

Sucrose 30 g 20 g 30 g 30 g 10 g 30 g 30 g

Agar 7 g 6 g 6 g 7g Phyto Agar 7 g 7 g Gelrite 3 g

*Media were prepared to pH 5.8 as a double concentrated stock and filter sterilized; the media were mixed 1:1 with double concentrated Agar (1.4%). The other media were adjusted to pH 5.8 and autoclaved.

Figure 5.4 Seedling from 7 days old Rapid-Cycling Brassica rapa


45

5.8.4 Plant tissue and protoplast culture in Nicotiana

tabacum

Seeds from Nicotiana tabacum cv. Petite Havana were surface sterilized with

70 % ethanol (v/v) for 1 min and then treated with 5 % (w/v) Dimanin C for

10 min. Seeds were washed three times for 10 min with autoclaved distilled water.

Tobacco seeds were geminated on modified B5 medium (Table 5.10) at 25 ºC with

photoperiod 16 h light (60 µmol/m2s) at 25 ºC followed by 8 h dark. After one

week, seedlings were isolated and growth during 3 weeks with the same growing

condition.

Tobacco regeneration was obtained from leaves on RMOP medium (Svab et al.,

1990) or from protoplast derived colonies as described elsewhere (Dovzhenko et

al., 1998). For tobacco protoplast isolation, leaves from 2-3 weeks old plants were

cut into approximately 1 mm stripes. For pre-plasmolysis, strips were incubated

for one hour in a 9 cm Petri dish containing F-PIN medium (Table 5.10), for cell

wall degradation, medium was discarded and changed for an F-PIN medium

containing 0.25 % cellulase R-10 and 0.25 % macerozyme R-10 (see 5.8.2). Cells

were further incubated in darkness at 25 C for 14-16 h.

Figure 5.5 Nicotiana tabacum leaf strips in F-PIN medium

The incubation mixture was passed through a stainless steel sieve (pore size,

100 m) and the filtrate was adjusted to 10 ml using F-PIN medium and then

overlayered with 2 ml of MMM medium (Table 4.8, osmolarity was adjusted to

550 mOsm). Followed by centrifugation for 10 min at 50 g intact protoplasts were


46

removed and adjusted to 10 ml with MMM medium. Counting and embedding of

protoplasts was realized as described before in section 5.8.2. Protoplasts were

embedded at a density of 6 X 104 pp/grids. Grids with embedded protoplasts

were placed upside down in a 6 cm Petri dish containing 10 ml of F-PCN medium

(Table 5.10). After 30 min of incubation, grids were transferred into a new Petri

dish (6 cm in diameter) with 2 ml of F-PCN medium. After protoplast derived

colonies were formed, grids were placed on 9 cm Petri dishes containing solid

RMOP medium. Regenerated shoots were rooted on hormone-free B5 mod

medium.

Table 5.10 Nicotiana tabacum tissue culture media

Components / l B5 mod

(Dovzhenko, 1998)

F-PIN

(Dovzhenko, 1998)

F-PCN

(Dovzhenko, 1998)

RMOP

(Svab et al., 1990)

MS Basal medium 4.4 g B5 Basal medium 3.1 g

Macro MS mod 10X 100 ml 100 ml Micro MS 100 X 10 ml 10 ml

NT Vitamins 100 X 10 ml B5 Vitamins 100 X 10 ml PC Vitamins 100 X 10 ml 10 ml

NH4- Succinat (2M)a 10 ml 10 ml MgSO4·7H2O 0.983 g MES 1.952 g 1.952 g

BAP (1mg/ml) 1 ml 1 ml 1 ml NAA (1mg/ml) 100 µl 100 µl 100 µl

Sucrose 20 g 130 g 20 g 30 g

Glucose 65 g

Agar 8.0 g 6 g

All media were adjusted to pH 5.8. B5 mod and RMOP were autoclaved and the other media were adjusted to 550 mOsm and sterile filtrated with 0.2 µm-Filter. a 2 M Ammoniumsuccinat was prepared according to (Dovzhenko et al., 1998): 23.6 g Succinic acid, 10.6 g NH4Cl, approximately 22.4 g KOH to pH 5.8 adjusted to 100 ml and the solution was sterile filtrated.

5.9 Plant transformation

5.9.1 PEG treatment of protoplasts

The polyethylene glycol (PEG) method for nuclear or plastid transformation

was performed as described previously (Koop et al., 1996).


47

Solutions:

Transformation medium: 1l 40 % PEG solution: 26 ml

MgCl2·6H2O 3,045 g Manitol 1.275 g MES 1.0 g Ca(NO3)2·4H2O 0.413 g Manitol ~ 85 g to 550 mOsm PEG-1500 10 g Distilled H2O to 1 l Distilled H2O to 26 ml mix to dissolve pH 5.6 and sterile filtrated. pH 9.75 and sterile filtrate d

Protoplasts from Arabidopsis thaliana (see 5.8.2) or tobacco (see 5.8.4) were isolated,

1 X 106 pp were suspended in 100µl of Transformation medium and subsequently

transferred to a 3 cm Petri dish made of glass. 50 µg of DNA, dissolved in 18 µl TE

pH 5.6, were mixed with protoplasts and 7 µl of F-PCN (tobacco) or PCA

(Arabidopsis thaliana). Immediately, 125 µl of PEG were added drop wise and

incubated for 7.5 min. Afterwards, 125 µl of F-PCN or PCA were added and

incubated for 2 min. Finally, 2,6 ml of F-PCN or PCA were added and protoplasts

were incubated for 12 h at 25 ºC in the dark. Protoplasts were embedded with

alginate as described previously in 5.8.2 (Arabidopsis thaliana) or 5.8.4 (tobacco).

After 7 days in liquid culture media, protoplast derived colonies were transferred

to regeneration media with appropriate antibiotics for the selection of plants.

5.9.2 DNA transfer using the particle gun method

For particle gun experiments, a standard protocol was used. Sterilization of

particle gun chamber, microcarrier launch assembly, rupture disc holder, target

shelf, macrocarrier holder, macrocarriers and stopping screens was performed

with 100 % EtOH. Rupture discs were sterilized individually by washing them in

75 % isopropanol for 5-10 s. Subsequently, all components were air dried in the

flow bench. Then, 60 mg of Gold particles (0.6 µm particle size from Bio-Rad,

USA) were suspended in 1 ml EtOH p.a. The gold particle suspension was

vortexed for at least 1 min and then 35 µl were added immediately into a new

sterile 1.5 ml Eppendorf tube. The gold was spun down at 13 000 rpm for 10 s and

the supernatant was removed. Working on ice, 230 µl sterile dH2O were mixed

with the gold pellet. Because the gold needs to be resuspended, this is best


48

achieved by a combination of vortexing and scraping from the wall with a Gilson

tip. After addition of 250 µl of 2.5 M CaCl2 (stored in aliquots at -20 ºC) and its

subsequent vortexing, 25 µl of DNA (1.0 µg/µl in water) were added. Once again,

the sample was mixed with a vortex for a few seconds, and 50 µl of 0.1 M

spermidine (aliquots -70 ºC) were added. After vortexing, the mixture was

incubated 10 min on ice and vortexed again every 2 min. The gold was spun down

for 1 min at 13 000 rpm, and the supernatant was removed. The gold was washed

2 times with 600 µl of 100 % EtOH p.a (between each step a centrifugation for 1

min was necessary). It is very important that the fine gold is removed from the

wall and that the pellet is broken up. Finally, the gold was resuspended in 72 µl of

100 % EtOH p.a and placed on ice.

Shooting parameters:

Pressure of helium: 1100 psi (7584.17 Kilopascal) Rupture discs: 900 psi (6205.2 Kilopascal) Distance rupture disc-macrocarrier: 8-10 mm Distance macrocarrier-stopping screen: 10 mm Distance stopping plate-leaf: 7 cm Vacuum: 27 – 28 inches Hg (91.4-94.8 Kilopascal)

5.4 μl of the DNA-coated gold suspension were applied to the middle of each

macrocarrier and air-dried completely. The macrocarrier was placed into the

macrocarrier holder. The rupture disc, the stopping screen and the macrocarrier

holder were deployed correctly in the vacuum chamber. The Petri dish containing

plant material was placed at the right position inside the chamber, the chamber

door was closed and the vacuum was turned on until 26-27 inches Hg. The

pressurized helium tank was open and bombardment was realized according to

Bio-Rad instructions. Then, plant material was removed and transferred into a

clime chamber for one or two days before selection.


49

5.10 Selection conditions

PEG in Arabidopsis and tobacco: After the initial 6-8 days of protoplast culture,

500 mg/l spectinomycin for tobacco or 20-40 mg/l spectinomycin for Arabidopsis

thaliana were included in solid regeneration media for the selection of colonies.

After bombardment: Plant material was transferred to selection medium

supplemented with antibiotic, 3 days after bombardment. Resistant colonies were

collected after 4-5 weeks of culture and then transferred to same fresh media:

Selection Tobacco Arabidopsis RCBr

Kanamycin 10 – 40 mg/l

Spectinomycin 500 mg/l 10 – 40 mg/l 10-40 mgl/l Phosphinothricin 1 – 7 mg/l Bialaphos 2 – 10 mg/l

5.11 Microscopy

In order to characterize putative plastid transformed lines, epidermal

peelings were prepared from young leaves using water. To analyze chlorophyll

autofluorescence (excitation: 543 nm, emission: 580 nm) and AmCyan fluorescence

(excitation: 543 nm, emission: 580 nm), samples were analyzed with a fluorescence

microscope from Leica Microsystems (DM 1000). Finally, to acquire better quality

pictures, a Leica TCS SP2 spectral laser-scanning confocal microscope (Leica

microsystems, Wetzlar, Germany) was employed.

Results

50

6 Results

The present work focuses on the establishment of a protocol for plastid

transformation in plant model organisms. Therefore, regeneration protocols in

Arabidopsis thaliana, Rapid-Cycling Brassica rapa and Nicotiana tabacum were

established, plant specific vectors with different insertion sites were constructed

and finally, a PEG (Polyethylene glycol) or biolistic transformation method was

used for the transformation of the plants.

6.1 Towards plastid transformation in Arabidopsis thaliana

For plastid transformation experiments in Arabidopsis thaliana different

experimental approaches were used: 1. A reliable regeneration protocol was

established. 2. Plant specific vectors for plastid transformation were constructed. 3.

Vectors with different fluorescence and selection markers were tested. 4. Two

different transformation methods were performed.

6.1.1 Regeneration from cotyledons in Arabidopsis thaliana

For regeneration experiments, cotyledon explants from 3-9 days old Arabidopsis

thaliana ecotype C-24 were used. As expected, shoot regeneration was reduced

dramatically in old explants (Figure 6.1-a) as described by Zhao et al., (2002). The

best regeneration was obtained with 3 days old cotyledons. However, 3 days old

seedlings are not easy to manipulate and to use in particle gun experiments. In this

work, 6-7 days old seedlings were used for the establishment of regeneration

protocols. Preculture of cotyledons on callus induction medium (CIM) for 2-4 days

(section 5.8.1) improved explant regeneration greatly in comparison with

cotyledon shoot regeneration Arabidopsis medium (SRAKN). It almost doubled the

regeneration efficiency from 45 % to 85 % (Figure 6.1-b).

Approximately 3 to 4 shoots were obtained from 6 days old explants after

incubation of the cotyledons on CIM for two days and subsequent incubation in

SRAKN medium for 3 weeks. Although silver nitrate is widely used to improve

Results

51

regeneration efficiencies in Brassicaceae, in our experiments the addition of silver

nitrate had a negative effect on regeneration. This effect was very severe in the

case of CIM/SRAKN in which regeneration efficiency was reduced from 85 % to

28 % (Figure 6.1-b).

Figure 6.1 Regeneration from cotyledons in Arabidopsis. (a) Effect of explant age on regeneration efficiency. (b) Influence of different media on shoot regeneration from cotyledons in Arabidopsis thaliana C-24 after three weeks on the indicated medium.

Exposure of regenerated shoots on a root induction medium for 10 days increased

root formation in greater than 90 %. These regenerated plants were able to mature

into fertile plants. Figure 6.2 shows the complete regeneration cycle starting from

cotyledons to fertile Arabidopsis thaliana C-24 plants.

Figure 6.2 Arabidopsis thaliana C-24 regenerated from cotyledon explants. (a) Seedlings growing on SCA germination medium. (b) Cotyledon on CIM medium for callus induction. (c) Shoot formation was induced on CIM/SIM media after 20 days. (d) Shoot formation was induced on SIM medium after 20 days. (e) Mature plants on MB medium for seed production. Bars: 3 mm.

Results

52

6.1.2 Regeneration of Arabidopsis thaliana from seed

explants

Regeneration from Arabidopsis thaliana C-24 using seeds as explants was already

described (Negrutiu et al., 1975), however, we improved the seeds regeneration

method. Media components are described on section 5.8.1. In summary, two

protocols were used, one protocol was based on Márton and Browse (1991) and

the other one based on PG derived media from Negrutiu et al., (1975).

Based on Márton and Browse method: Arabidopsis thaliana C-24 seeds were

germinated on ARMI medium (Table 5.6). After 10-12 days on ARMI medium,

callus formation was analysed and two characteristic types of seedlings were

observed. Seedlings were either short with abundant callus (Figure 6.3-a) or

seedlings were longer with a small callus formation between hypocotyl and roots

(Figure 6.3-b). After two weeks on ARMI medium, almost 90 % of seedlings

produced calli. Those calli were transferred to fresh medium for additional two

weeks. Calli from ARMI medium were abundant and compact as described before

(Azhagiri and Maliga, 2007). For shoot induction, four weeks old calli were

transferred to SRAKN, SRA2N and ARMIIc regeneration media (Table 5.7).

After 20 days on ARMIIc medium, just small shoots were obtained in

approximately 50 % (Figure 6.3-c). Best results were obtained when calli were

transferred to SRAKN medium with up to 83 % of regeneration (Table 6.1).

Figure 6.3 Callus induction on ARM media. (a) Short seedling with abundant calli, after 12 days on ARMI medium. (b) Long seedling with small calli (arrow) between roots and hypocotyl after 12 days on ARMI medium. (c) Abundant calli after 20 days on ARMIIc medium. Bars: 3 mm

Results

53

Additionally to ARM media, Arabidopsis thaliana C-24 seeds were germinated on

PG derived media (based on Negrutiu et al., 1975). Therefore, seeds were placed

on PG1KD, PG20KD, PG1KN and PG20KN media (Table 5.6) either in the

darkness or in the light. Seeds germinated to give small seedlings within 3-4 days.

As described by Huang and Yeoman, (1985), callus was initiated from hypocotyl

and from roots of the seedlings and became visible after 12-15 days (Figure 6.4-j).

Around 80-95 % of the plated seeds produced calli on PG derived media as

described in Table 5.1. Seedlings on PG derived media with Ara or B5 Vitamins,

usually were translucent, however, in presence of Cocktail 20, seedlings were

opaque (Figure 6.4). After 3-4 weeks on PG derived media, callus from seeds were

almost 30 times bigger.

Seed derived callus could be induced on all media, without distinction between

use of auxins or vitamins. Callus induced on PG derived media containing NAA

(8 mg/l NAA) looked hairy, rather compact, and had root-like formations on its

surface as described previously (Negrutiu et al., 1975).

After one month on callus induction media, calli were transferred to SRAKN,

SRA2N and ARMIIc regeneration media (Table 5.7). One week on regeneration

media was sufficient for shoot regeneration (Figure 6.4). Seeds which were

germinated under light gave the best results. Shoots were quantified and results

are presented in Table 6.1. In summary, best regeneration efficiency was achieved

with the following combination of media: four weeks on PG20KN and subsequent

transfer to SRA2N medium (up to 90 %) or four weeks on PG20KD and transfer to

SRA2N medium with up to 93 % or SRAKN medium with up to 97 % regeneration

(Table 6.1).

Table 6.1 Regeneration from seed derived callus

Calli media Calli induction (%) Shoot regeneration (%)

SRAKN SRA2N ARMIIc PG1KN 85 ± 5 50 ± 5 50 ± 5 --- PG1KD 90 ± 5 81 ± 5 66 ± 1 87 ± 1

PG20KN 85 ± 5 20 ± 5 85 ± 5 --- PG20KD 90 ± 5 97± 1 92 ± 1 90 ± 2

ARMI 85 ± 5 83 ± 10 80 ± 7 50 ± 10

Results

54

Regeneration obtained with the combination of PG1KN–SRAKN or SRA2N was

not always reliable (Table 6.1). Low regeneration (approximately 20 %) was

obtained with PG20KN–SRAKN combination media, meaning that many roots

were formed (Figure 6.5-f). However when calli was transferred from PG20KN to

SRA2N medium, approximately 85 % of all calli were able to produce shoots. With

this combination (PG20KN-SRA2N) 2-4 shoots per explant were obtained (Figure

6.5-e).

Callus induced on media containing 2,4-D as auxin grow rapidly and presented a

better friability. Regeneration between 3 to 7 shoots per explant were obtained

either with cocktail 20 or Ara vitamins (Figure 6.4). Although, number of shoots

was similar in all cases, best regeneration results were achieved with PG20KD-

SRAKN with up to 97 %of regeneration. This PG20KD medium contains cocktail

20 a mixture of nitrogen compounds and vitamins (Mere-Villanueva and Vazquez-

Alejandro, 2003). Addition of cocktail 20 in tissue cultures of carrot (Daucus carota)

improved significantly the regeneration efficiencies (Rosales-Mendoza, 2007). In

Arabidopsis thaliana up to 97 % of regeneration efficiencies was obtained. This

represent the number of seed derived calli producing shoots. If the numbers of

shoots per explant are included, we obtained regeneration efficiencies between 500

to 700 % (Figure 6.4).

Figure 6.4 Indirect shoot organogenesis from seed derived calli in Arabidopsis thaliana C-24. Combination PG20KD/SRAKN media. Bar: 5 mm.

Results

55

Shoots were transferred to a root induction media (Table 5.7) or to a hormone free

medium (SCA, see table 5.6). Healthy fertile plants were obtained from all media

combination. Figure 6.5-m shows an example from healthy fertile plant obtained

from seeds derived calli.

Regeneration capacity from seed derived calli remained stable for at least 6 weeks

old calli (data not shown).

Figure 6.5 Shoot regeneration from seed derived callus of Arabidopsis thaliana C-24. (a, d, g, j) 10 days old seedlings on PG1KN, PG20KN, PG20KD and PG1KD respectively. (b, e, h, k) regeneration from one month old calli, after 12 days on SRA2N and (c, f, i, l) on SRAKN medium (m) Mature plant growing on SCA medium after 3 weeks. Bars: 1 mm

Results

56

6.1.3 Arabidopsis thaliana protoplast culture and

regeneration

After establishing a protocol for the regeneration of plants from cotyledons and

seeds the next step was to regenerate plants from cotyledon protoplasts.

Regeneration from Arabidopsis cotyledon protoplasts was reliable using a modified

method from our group (Dovzhenko et al., 2003). The main modifications

consisted in seeds sterilisation with Cl2 gas, germination with intensive light (130-

150 μmol/m2s1), modification of Protoplast culture medium (PCA) and finally

addition of Cefotaxime (50 mg/l) on germination and on PCA medium to avoid

contamination. Dovzhenko et al., used Dicamba and NAA as phytohormones in

the presence of casein. In this work presence of casein was avoided and Cytokinin

2-ip was used instead auxin Dicamba. Platting efficiencies were increased up to

40-60 %, almost twice as reported by Dovzhenko et al., (2003).

In the present work 2,4-D (2,4-Dichlorophenoxyacetic acid) (1mg/l), a synthetic

auxin which supports dedifferentiation and cell division in plant tissues was

tested in PCA medium. This also increased platting efficiencies, however,

protoplast derived colonies were not able to produce many shoots on SRAKN or

SRA2N media (data not shown). Normally, first cell divisions were found after 1

or 2 days on PCA medium (Figure 6.6).

Results

57

5-7 days after protoplast isolation, protoplast derived colonies became visible with

the naked eye (Figure 6.7). Grids containing 7-8 days old protoplast derived

colonies were transferred to shoot regeneration medium. The best regeneration

medium contains Kinetin and NAA as phytohormons (SRAKN). First shoots were

observed after 5-6 days (Figure 6.7 b-c). After 15-20 days on SRAKN medium,

more than 120 shoots per grid were obtained (Figure 6.7-f).

(a)

(b)

(c)

(d)

Figure 6.6 Arabidopsis thaliana C-24 cotyledon protoplasts (a) Cotyledon protoplasts, one day after isolation. Cotyledon protoplasts cell divisions three (b), five (c), seven (d) days after protoplast isolation. Bars: 10 µm

Results

58

SRAKN medium revealed the best regeneration efficiencies; however,

regeneration was extremely fast (just five days). To support the selection of

transformed cells, a regeneration protocol with a prolonged callus phase might be

required. Therefore, SRAKN medium was modified by the addition of 0.25,

0.5 mg/l or 1 mg/l of 2,4-D. SRA2N medium was used at same time and 7 days

old protoplast derived colonies were transferred to those media. Results are

shown on Figure 6.8. As expected, addition of 2,4-D to SRAKN medium allowed

the induction of calli from protoplast derived colonies after two weeks. Addition

of 0.25 mg/l of 2,4-D was sufficient to produce calli as shown on Figure 6.8 (a).

SRA2N and SRAKN + 0.25 m/l 2,4-D were the best option to produce calli from

protoplast derived colonies, because competence to produce shoots was

maintained and fertile plants were obtained.

Figure 6.7 Shoot formation from protoplast derived colonies in Arabidopsis thaliana C-24. (a, d) 9 days old protoplast derived colonies on Regeneration media. (b, c, e) First shoots from protoplast derived colonies after 5 days on SRAKN medium. (f) Grid containing shoots from protoplast derived colonies after 20 days. Bars: 2 mm

Results

59

Figure 6.8 Callus induction from Arabidopsis thaliana C-24 protoplast derived colonies. (a) Grid containing calli and shoots from protoplast derived colonies after three weeks on SRA2N medium. (b, c, d) Grids containing calli from protoplast derived colonies after three weeks on SRAKN media containing 0.25 mg/l; 0.5 mg/l and 1 mg/l 2,4-D respectively. Bars: 5 mm

Once shoots from protoplast derived colonies were obtained, those shoots were

transferred to phytohormone-free medium or to root induction medium

supplemented with auxins (Table 5.7). Shoots from protoplast derived colonies

were competent to form roots (80-90 %) in almost all media, including medium

free of phytohormones (Figure 6.9). To produce seeds, the plants were either

cultured on phytohormone-free medium, root induction medium or transferred to

soil and were grown in the greenhouse.

Figure 6.9 Root induction from shoots of protoplast derived colonies in Arabidopsis thaliana C-24. (a) Root induction after two weeks on ½MS medium free from phytohormones. (b) Root induction on medium RIMI containing IBA as auxin. (c) Root induction on medium RIMII containing IBA and NAA as auxins. Bar: 1 cm

Results

60

6.1.4 Evaluation of the sensitivity of Arabidopsis thaliana

protoplast derived colonies to selection agents

The next step on the road to stable plastid transformation was the optimization

of selective conditions. In this work, kanamycin, spectinomycin, bialaphos and

phosphinothricin selection conditions for Arabidopsis thaliana plastid

transformation, were tested. Four different genes were used as selection marker:

nptII gene encodes neomycin phosphotransferase II (Carrer et al., 1993), the aphA6

gene encodes a 3´aminoglycoside phosphotransferase type VI (Huang et al., 2002)

both genes confer a resistance to Kanamycin; the aadA gene encodes

aminoglycoside 3´transferase (AADA) (Svab and Maliga, 1993) conferring

resistance to spectinomycin and streptomycin and finally the bar gene encodes a

phosphinothricin acetyl transferase (PAT) enzyme (Lutz et al., 2001) and confers

resistance to the herbicides phosphinothricin and bialaphphos. To determine the

optimal antibiotic or herbicide concentration for the inhibition of callus growth

and shoot induction, grids containing protoplast derived colonies were transferred

to regeneration medium containing kanamycin (0, 20, 25, 30 or 40 mg/l),

spectinomycin (0, 5, 10, 15 or 20 mg/l) or phosphinothricin (0, 2.5, 5, 7, 10 mg/l)

(Figures 6.10-6-11).

Figure 6.10 Effect of spectinomycin (Spec), kanamycin (Kan) on shoot regeneration from protoplast derived colonies in Arabidopsis thaliana C24. Protoplast derived colonies after 20 days on SRAKN media containing spectinomycin 0 (a), 5 (b), 10 (c), 15 (d) and 20 € mg/l. Protoplast derived colonies after 20 days on SRAKN media containing Kan 0 (f), 20 (g), 25 (h), 30 (i) and 40 (j) mg/l. Shoots were detected in media containing 20 mg/l of Kan with inhibition of regeneration on media containing Kan 25 mg/l. Bars: 5 mm

Results

61

Addition of 10 mg/l of spectinomycin to SRAKN medium was sufficient for

inhibition of shoot regeneration. On SRAKN medium containing 5 mg/l

spectinomycin, two shoots were obtained after 20 days. After 25 days on SRAKN

containing 15 mg/l spectinomycin, shoots were obtained, indicating that

spectinomycin was not stable and that for successful selection fresh antibiotic is

required. Addition of 25 mg/l of kanamycin to SRAKN media was necessary to

ensure inhibition of the regeneration from protoplast derived colonies.

The inhibition of regeneration strongly depends on the presence of the herbicide

phosphinothricin (PPT). In the presence of 2.5 mg/l PPT in SRAKN medium 10 to

15 protoplast derived calli were formed, however this number could be decreased

to one or two calli per grid by the addition of 5 – 10 mg/l PPT (Figure 6.11).

Higher concentrations of PPT (15 mg/l) were highly toxic for protoplast derived

colonies. Therefore, addition of 2.5 or 3 mg/l of PPT on SRAKN medium seem be

sufficient for selection of protoplast derived colonies, in plastid transformation

experiments.

6.1.5 Construction of Arabidopsis thaliana plastid

transformation vectors

To allow stable integration into the Arabidopsis plastome, vectors with markers

flanked by Arabidopsis plastid sequences were required. Because insertion of

sequences in plastid transformation occurs by homologous recombination (Staub,

et al., 1992), it was necessary to choose Arabidopsis plastome sequences flanking the

selected integration site.

Figure 6.11 Effect of phosphinothricin (PPT) on shoot regeneration from protoplast derived colonies in Arabidopsis Protoplast derived colonies after 20 on SRAKN medium containing PPT 0 (a), 2.5 (b), 5 (c), 7.5 (d) and 10 (e) mg/l. Bars: 5 mm

Results

62

Homologous fragments (INSL and INSR) flanking the targeted insertion point

between rbcL (Nucleotide positions in the NCBI database: 54958-56397) and accD

(Nucleotide positions in the NCBI database: 57075-58541) genes were amplified

using total Arabidopsis thaliana C24 DNA as template and the PCR primers

AraRub-XbaI (ACGTCTAGAGGATCTGCGAATC) and AraccD-BamHI (AATT

TAATGGGATCCGCAGAG). The 2.5 Kb PCR product was digested with SpeI-

BamHI enzymes and this fragment was ligated into pUC18 vector using XbaI-

BamHI sites. This new vector containing rbcL and accD from Arabidopsis thaliana

C24 was named pC24 vector. pC24 vector was sequenced and a substitution C-G

was detected in the accD gene. A C-G substitution represents a change from

Threonine to Serine in the accD gene (Appendix 1).

Additional vectors containing other recombination regions in the Arabidopsis

thaliana plastome were also used in this work. pSTS4 vector with flanks for

insertion between trnfM and trnG genes was obtained from Prof. Ralph Bock

(Max-Planck-Institute of Molecular Plant Physiology). pBA vector with flanks for

insertion between trnV-rps12/7 was obtained from Christian Stelljes. The insertion

sites from the vectors in Arabidopsis thaliana plastome are shown on Figure 6.12.

In total three different insertion sites were chosen to allow homologous

recombination in the Arabidopsis thaliana plastome (Figure 6.12)

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63

In the present work visual and selective markers were chosen, as visual

fluorescence markers, three markers were used: DsRed (Red Fluorescent Protein),

GFP (Green Fluorescent Protein) and AmCyan (Cyan Fluorescent Protein). With

the help of visual markers transformed tissue can easily be selected (Khan and

Maliga, 1999). As selection marker four different genes were used: aadA, nptII,

aphA6 and bar genes (section 6.1.4). The mode of how fluorescence or selection

markers (with insertion sites) were combined is presented in Figure 6.13

Figure 6.12 Gen map of Arabidopsis thaliana chloroplast. Genes shown on the inner circle are transcribed clockwise, and those on the outside counter-clockwise. Genes with related functions are shown with the same color. The genome shows two copies of the inverted repeat region (IR) separating the large (LSC) and small (SSC) single copy regions. Arrows show insertion position for integration of foreign genes with the name of the vector containing those regions. The map was created using “OrganellarGenomeDRAW” software developed by M. Lohse and O. Drechsel in the Bock laboratory.

Results

64

Once vectors with insertion sites in the Arabidopsis plastome were obtained,

cassettes containing selection or fluorescence markers were constructed. In order

to achieve high expression in plastids, the Prrn tobacco 16S-rRNA (Svab and

Maliga, 1993), in addition to the 5’UTR of gene 10 from bacteriophage T7 (T7g10)

and MASIS sequences (Herz et al., 2005) were cloned in front of the AmCyan and

DsRed genes. The 3´UTR Tprl32 was cloned at the end of fluorescence markers.

A new vector containing DsRed was cloned in the following way: the DsRed gene

was cut out with NcoI-XbaI from plasmid pGJ1425, kindly provided by Angela

Dietzmann (Jach et al., 2001) and was ligated to the 3´ UTR regulatory region

(Trpl32) of the p546 vector. The cassette DsRed::Trpl32 was cut out with NcoI-XhoI

and was ligated with pUC18-Prrn vector and restricted with the same enzymes.

The new pUC18-DsRed vector (Figure 6.14-a) was used in the next steps. A Petri

dish with E. coli XL1 Blue colonies (transformed with pUC18-DsRed vector), were

observed under Fluorescence SteREO Lumar. V12 (Fig 6.14-c).

For the creation of plastid transformation vectors containing AmCyan

fluorescence marker, the AmCyan gene from pAmCyan vector (Clontech®) was

cut out with XbaI, and the fragment was further partially digested with NcoI. This

Figure 6.13 Plastid transformation vectors in Arabidopsis thaliana. 1. Homologous flanks in Arabidopsis plastome, vector name is indicated under bracket. 2. Selection or fluorescence marker was cloned between homologous flanks. 3. Selection marker was cloned outside of homologous flanks. 4. Name of plastid transformation vector is indicated.

Results

65

fragment was ligated with plasmid pUC18-DsRed, which has been digested with

the same restriction enzymes combination. The new pUC18-AmCyan vector was

sequenced and the expression of AmCyan was detected in E. coli XL1 Blue

colonies under Fluorescence SteREO Lumar. V12 (Fig 6.14-d).

The vector pC24-GFPbar with the Arabidopsis rbcL-accD flanks containing

PrrnT7g10-MASIS::gfpbar::Trpl32 cassette was kindly provided by Peter Wehmann

(AG Koop, LMU). Expression of GFP was also detected under UV (Figure 6.14-e).

Figure 6.14 Fluorescence markers for plastid transformation. (a) Schematic presentation of cloning steps of a DsRed vector for plastid transformation. (b) Schematic presentation of cloning steps of an AmCyan vector for plastid transformation. Restrictions sites for cloning steps are shown. (c, d) Colonies from E. coli XL1 Blue showing DsRed and AmCyan fluorescence, colonies were observed under Fluorescence SteREO Lumar. V12. (e) Colonies from E. coli XL1 Blue expressing GFPbar, observed under UV lamp. Bars: 2 mm.

Results

66

Figure 6.15 Schematic presentation of cloning step of a pSTS-aadA vector. Restriction sites for cloning steps are shown. Hatched boxes represent Arabidopsis homologous flanks.

pSTS-aadA vector was generated by ligating Prrn::aadA::TrbcL (NotI-XbaI) from

pKCZ-aadA vector into the NotI-XbaI of pSTS4 vector (Figure 6.15)

pSKD (pSTS-DsRed-aphA6) vector was cloned by ligating the cassette Prrn-T7g10-

MASIS::DsRed::Trpl32 (SalI-XhoI) from pUC18-DsRed into the Sal I site of pSTS4

vector. An aphA6 cassette in the backbone of pSTS-DsRed vector was cloned by

digestion with AviII and once vector was linearized, it was ligated to an

PpsbA::aphA6::TrbcL (SmaI) cassette obtained from pGEMaphA6 vector (kindly

provided by Dr. Lars Scharff AG Koop, LMU). Kanamycin resistant pink colonies

were chosen for further characterization (Figure 6.16).

Figure 6.16 Schematic presentation of cloning steps of a pSKD vector. Restrictions sites for cloning steps are shown. Hatched boxes represent Arabidopsis thaliana homologous flanks

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pSAD (pSTS-DsRed-aadA) vector was obtained by replacing aphA6 cassette in

pSKD vector for aadA cassette, pSKD vector was digested with NsbI-MluI enzyms,

vector was purified and ligated to an PpsbA::aadA::TrbcL cassette from pUC18-

aadA vector digested with MluI-NsbI enzyms. Ligation of those fragments was

transformed and spectinomycin resistant pink colonies were characterized and

sequenced (Figure 6.17).

pSAA (pSTS-AmCyan-aadA) vector was generated in two steps, first Prrn-T7g10-

MASIS::AmCyan::Trpl32 cassette (PstI-XhoI) from pUC18-aadA vector was cloned

into PstI-XhoI sites of pSTS4 vector. An Prrn-T7g10-MASIS::AmCyan::Trpl32

(BsrGI-XhoI) cassette from pSTS-AmCyan vector was cloned in BsrGI-XhoI sites of

pSAD vector. Resistant blue colonies were further characterized and the vector

was sequenced (Figure 6.18).

Figure 6.18 Schematic presentation of cloning steps of a pSAA vector. Restrictions sites for cloning steps are shown. Hatched boxes represent Arabidopsis homologous flanks.

Figure 6.17 Schematic presentation of cloning steps of a pSAD vector. Restriction sites for cloning steps are shown. Hatched boxes represent Arabidopsis homologous flanks

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pC24-aadA vector was obtained by ligating Prrn::aadA::TrbcL (SmaI-Eco47III)

cassette from pKCZ-aadA vector into the Bst1107I site of pC24 vector. pC24 vector

contains rbcL and accD flanks from Arabidopsis thaliana C-24. (Figure 6.19)

pC24-nptII vector was obtained by replacing aadA (XhoI-SpeI) cassette from pC24-

aadA vector for PNG1014::nptII::TrbcL (XhoI-SpeI) from pKCZ-nptII vector.

Kanamycin resistant colonies were characterized and sequenced (Figure 6.20).

Figure 6.19 Schematic presentation of cloning step of a pC24-aadA vector. Restrictions sites for cloning steps are shown. Hatched boxes represent Arabidopsis thaliana homologous flanks.

Figure 6.20 Schematic presentation of cloning step of a pC24-nptII vector. Restrictions sites for cloning steps are shown. Hatched boxes represent Arabidopsis thaliana homologous flanks.

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6.1.6 Particle gun and PEG-based plastid transformation

experiments in Arabidopsis thaliana

After the establishment of regeneration protocols in Arabidopsis thaliana and the

generation of appropriate vectors particle gun and PEG mediated plastid

transformation were realized with Arabidopsis thaliana plastome specific vectors

(Figure 5.9). Gold particles (0.6µ) covered with desirable DNA were used for

particle gun experiments as described in section 5.9.2. Grids containing 7-10 days

old protoplast derived colonies or 20 days old seed derived calli from Arabidopsis

thaliana C-24 were used as plant material. Around 6 to 9 grids were bombarded

with one vector each experiment. Two days after bombardment, plant material

was transferred to a selective regeneration medium as described on 6.1.4. In total,

43 independent particle gun experiments with 338 single bombardments were

executed with vectors containing aminoglycoside resistance genes (Table 6.2).

However, only some putative transplastomic lines could be obtained.

Particle gun experiments were performed with vectors containing DsRed as

fluorescence marker and aadA or aphA6 as selection marker in the backbone of the

vector (pSKD and pSAD vectors). After two transformation experiments with

pSKD vector, 4 putative transformant lines were obtained, with pSAD vector 8

putative lines were obtained. To detect DsRed expression and the presence of the

aphA6 gene, fluorescence microscopy and PCR were used, respectively. All

putative analysed lines were negative for the aphA6 gene and for the DsRed

fluorescent protein (Figure 6.21).

Figure 6.21 Arabidopsis thaliana plastid transformation experiments with pSKD vector. (a) Putative SKD line growing on SRAKN medium containing 20 mg/l kanamycin. (b) Results from putative lines analysed by PCR to detect the presence of the aphA6 gene (658bp). pSKD vector was used as postive control (+) and water as negative control (-) Lambda Eco 47I was used as a marker (M). Agarose gel 1 %. Bar: 5 mm

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Table 6.2 Particle gun experiments with aminoglycoside resistance genes

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DsRed gene was replaced with AmCyan gene as fluorescence marker for plastid

transformation experiments with pSAA vector introducing a new fluorescence

marker for plastid transformation (Wenck et al., 2003). The pSAA vector contains

an AmCyan cassette between Arabidopsis thaliana flanks and an aadA cassette on

the backbone of the vector (Table 6.2).

In general 74 grids containing protoplast derived colonies from Arabidopsis were

bombarded in 8 independent particle gun experiments with pSAA vector.

pSAA vector brought a higher number of putative transformants. In one

experiment around of 65 spectinomycin resistant shoots were obtained. However,

most putative lines (40 shoots) were obtained from two grids which were

transferred to SRAKN medium (Table 5.7) without spectinomycin after 20 days.

Figure 6.22 shows a putative line. Shoots from candidate lines were separated and

approximately 20 shoots were transferred to regeneration medium containing

40 mg/l of spectinomycin. Those lines became white and died after three weeks

under selective conditions. The rest of the candidate lines were further grown on

antibiotic free media, and healthy fertile plants were obtained (Figure 6.22-e).

Putative lines were analysed by fluorescence microscopy to detect AmCyan

expression and by PCR to amplify the AmCyan or the aadA genes. PCR was

negative in all analysed lines and no fluorescent line could be detected.

To further analyse the putative lines, T1 plants were germinated on SCA medium

containing 40 mg/l of spectinomycin, however, they only produced white

seedlings (data not shown).

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Figure 6.22 Arabidopsis thaliana plastid transformation experiments with pSAA vector. (a) Grid containing protoplast derived colonies bombarded with pSAA vector. First shoots were obtained after 12 days on SRAKN medium containing 20 mg/l spectinomycin. (b-c) Close-up from a spectinomycin resistant shoot. (d) SAA derived line growing on free antibiotic SRAKN medium after two weeks. € SAA derived lines growing on SCA medium production seeds. Bars: 5 mm

In summary, 203 plastid transformation experiments were performed with vectors

containing different selection markers in the backbone of the plasmid (pSAD,

pSKD and pSAA). Although, the number of putative transformants was huge, all

analysed lines were negative. In order to produce a callus phase before

regeneration, medium SRA2N (Table 5.7) was used. In addition vectors containing

selection markers between insertions flanks were used for bombardment

experiments. A summary of particle gun based plastid transformation

experiments with aminoglycoside selection markers and herbicide markers is

shown on Table 6.3.

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Table 6.3 Summary of particle gun based plastid transformation experiments in A. thaliana C-24.

As an alternative approach to the particle gun experiments, the PEG mediated

transformation was used in parallel. The PEG transformation method is well

established in chloroplast transformation and has been widely used in transient

nuclear transformation in Arabidopsis thaliana protoplasts (Golds et al., 1993).

However, most protoplasts died after PEG treatment and thus cannot be used for

regeneration experiments. To establish a PEG transformation protocol which

allows the stable transformation of protoplasts, the PEG concentration was

reduced and protoplasts were incubated in PCA medium. In the present work

protoplasts were treated either with 20 % or with 40 % PEG1500 in the presence of

pGFP vector (kindly provided by PD Dr. Cordelia Bolle) (50 µg) as described in

section 5.9.1. GFP gene is under the 35S promoter (Promoter from cauliflower

mosaic virus (CaMV) and the nos terminator (nopaline synthase). GFP is

expressed in cytoplasm from protoplasts.

Two days after PEG transformation, transient GFP expression was detected in the

cytoplasm by fluorescence microscopy meaning that the PEG method is working

(Figure 6.23). Subsequently, protoplast derived colonies were cultured and

regenerated on non-selective PCA and SRAKN medium.

Vector Insertion in Arabidopsis

Selection Marker

Fluorescence Marker

Particle gun experiments

Putative lines

PCR

pSAD trnfM-trnG aadA DsRed 48 8 (-) pSKD trnfM-trnG aphA6 DsRed 13 4 (-) pSAA trnfM-trnG aadA AmCyan 74 89 (-) pSTS-aadA trnfM-trnG aadA --- 80 --- --- pC24-aadA rbcL-accD aadA --- 23 30 (-) pC24-nptII rbcL-accD nptII --- 100 29 (-) pC24-GFPbar

rbcL-accD bar GFP 70 -- --

pBA-GFPbar rps12/7-trnV bar GFP 95 -- --

Total 503 126

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Two plastid transformation experiments with pC24-nptII and pSTS-aadA vectors

were realized, but all protoplast derived colonies died under selective conditions.

In addition ten PEG mediate plastid transformation experiments using the pC24-

gfpbar vector were realized. In all cases no resistant plants were obtained.

Figure 6.23 PEG transformation from Arabidopsis thaliana C-24 with a pGFP vector. (a) Protoplast under DIC. (b) Chlorophyll (c) GFP filter. Protoplast were analyzed by confocal laser scanning microscopy. Bars: 10 µm

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6.2 Towards plastid transformation in Rapid-Cycling Brassica rapa

Rapid-Cycling Brassica rapa (RCBr) represents a new model system in plant

biology (Williams and Hill, 1986; Musgrave, 2000). RCBr are small in size

(approximately 20 cm), require a minimum time from seed to flowering (14-17

days), are highly female fertile and life cycle is short (28 to 35 days). We first

established a RCBr plant regeneration protocol focusing on lines C1 and B3. B3

lines are wild type RCBr and C1 lines are a anthocyanin less mutant, anl/anl and

require cross pollination. Then, plant specific vectors for plastid transformation

with AmCyan fluorescence marker and the aadA gen which confers resistance to

spectinomycin and streptomycin antibiotics, were constructed and RCBr was

bombarded with the help of a particle gun.

6.2.1 Plant regeneration from Rapid-Cycling Brassica rapa

To develop a suitable regeneration protocol different media with two different

explants were used (Table 6.3). First, two protocols with RCBr cotyledons as

explants and two different media were utilized for plant regeneration (Teo et al.,

1997; Cogbill et al., 2010). However, no shoots could be obtained (Figure 6.24) (data

for Cogbill et al., 2010 method, are not shown).

Figure 6.24 Callus production from cotyledons of Rapid-Cycling Brassica rapa (RCBr)- C1(a) 7 days old cotyledons from RCBr-C1 on MDN medium. (b) Cotyledon derived calli from RCBr-C1 after three weeks on MDN medium. Bars: 5 mm

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Table 6.4 Summary from regeneration experiments in Rapid-Cycling Brassica rapa

Protocol α (Chen and Celio, 2000)

β (Teo et al.,

1997)

γ (Jonoubi et al., 2005)

Explant Hypocotyls Cotyledons Hypocotyls Preculture ---- ---- 2 days on MD1 medium Calli 3 weeks on MDI medium ---- 7 days on BD1 medium Regeneration 3 weeks on MZI medium 3 weeks on 3 weeks on SIM medium Regeneration 3 weeks on MZI medium MDN medium 3 weeks on SIM medium

Efficiences C1 20-50 % 0 50-80 %

Efficiences B3 20-40 % 0 20-50 %

In a second set of experiments, hycototyls were utilized as explants. Protocols

were originally described in Brassica napus (in this work named as “α” Chen and

Celio, 2000and “γ” (Jonoubi et al., 2005)). Regeneration efficiency was about

20-50 % in protocol “α”. However, protocol “γ” was even better with efficiencies

of 50 -70 % (Figure 6.25). In all cases, shoots and fertile plants could be achieved.

Figure 6.25 Regeneration from Rapid-Cycling Brassica rapa C1. (a) 7 days old cut seedling from Rapic-Cycling Brassica rapa (b) Hypocotyl segments on MD1 preculture medium. (c) Hypocotyl segments on BD1 callus induction medium. (d) First shoots on hypocotyl segments after four weeks on SIM medium to induce shoot production. (e) Shoot after one week on hormone-free medium. (f, g) Shoots after two weeks on root induction medium. (h) Mature plants on RS medium for seed production. Bars: 4 mm

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6.2.2 Particle gun experiments for plastid transformation in

Rapid-Cycling Brassica rapa

For plastid transformation in Rapid-Cycling Brassica rapa a vector was

constructed, with insertion sites between the trnV-rps12/7 genes. Fragments were

amplified using Rapid-Cycling Brassica rapa (B3) total DNA as template and the

PCR primers BrFw (AATTGAGCTCGTTCTATTCG) and BrRw

(ATTGTCGACTTAC TGCCACT). The PCR product from RCBr was cloned into

pJET® vector. One out of four clones was sequenced and no errors were detected.

The PCR product was 100 % identical in sequence to Brassica rapa subsp.

Pekinensis chloroplast sequence (NCBI: DQ231548.1/GI:85816402). Brassica rapa

sequence was subcloned (SacI-SalI) in pUC19 vector.

pUC19-Br vector containing RCBr insertion sequence was linearized with Eco RV

enzyme and dephosphorylated with SAP enzyme. The linearized fragment was

ligated with an AmCyan cassette (PpsbA::AmCyan::Trlp32) which came from a

digestion of pICF-BB-AmCyan vector (Torabi, 2009) with XhoI-SmaI. The XhoI site

was removed by filling-in the site with the Klenow fragment of DNA polymerase.

XL1 Blue competent cells were transformed with the ligation mixture. Positive,

blue fluorescing colonies were characterized (Figure 6.26).

In order to obtain a vector containing an aadA cassette, pUC19-Br-AmCyan vector

was digested with ScaI-PstI and the fragment containing AmCyan cassette was

cloned into the ScaI-PstI sites from pUC18 vector. pUC18-Br-AmCyan vector was

digested with KpnI-SalI and ligated to a KpnI-XhoI fragment containing an aadA

cassette (Prrn::aadA::TrbcL) from pC24-aadA vector. XL1 Blue competent cells

were transformed with this ligation mixture and colonies growing on LB-Spec

100mg/l medium were obtained. pBr-AmCyan-aadA vector was characterized

and purified vector was used to particle gun experiments on RCBr plants.

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Figure 6.26 Schematic presentation of cloning step of a pBr-AmCyan vector. Restriction sites for cloning steps are shown. Hatched boxes represent Rapid-Cycling Brassica rapa homologous flanks.

After the development of a plant regeneration protocol and the construction of a

specific plastid transformation vector, particle gun experiments were performed.

Hypocotyls from RCBr were cut from around 70-80 plants and transferred to a

preculture medium two days before bombardment experiments.

Two days after particle gun bombardment, hypocotyls were transferred to a BD1

callus induction medium (Table 5.9) containing 20 mg/l spectinomycin, for 7 days

and subsequently were transferred to a SIM medium containing 20 mg/l

spectinomycin. Figure 6.27 shows hypocotyls prepared for particle gun

experiments and the effect of spectinomycin on shoot regeneration in RCBr

hypocotyl explants. In summary 15 mg/l spectinomycin was sufficient to inhibit

shoot regeneration.

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In total, 12 particle gun experiments were done, using hypocotyls from RCBr as

explants. Eight experiments were realized using pBr-AmCyan-aadA vector. In

addition, four experiments were performed with pC24-AmCyan-aadA (with

Arabidopsis thaliana flanks). From approximately 9,000 explants only one green

callus was obtained. However, this green callus bleached after 15 days on SIM

containing Spec 15 mg/l.

Figure 6.27 Effect of spectinomycin (Spec) on shoot regeneration from hypocotyls in RCBr. Hypocotyls on SIM medium containing Spec 0 (a) 5 (b), 10 (c), 15 (d), 20 (e) mg/l. Hypocotyls on MD1 medium for particle gun experiments. Bars: 3 mm.

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6.3 AmCyan as visual marker for plastid transformation

in Nicotiana tabacum

AmCyan fluorescent protein (from Anemona majano) represents a new

alternative as visual marker in plant biotechnology (Wenck et al., 2003). One

advantage of AmCyan is its brightness relative to eGFP fluorescent protein and

another one is its resistance to photobleaching. So far, however, only nuclear

transformation has been achieved. In the present work an AmCyan gene with

regulatory elements for plastid transformation was used in Arabidopsis thaliana and

RCBr. To determine whether the AmCyan cassette can be transcribed and

expressed in plastids, Nicotiana tabacum cv. Petit Havana was transformed as a

control. A tobacco pKCZ (Zou et al., 2003) derived vector, containing an AmCyan

cassette (PrrnT7g10-MASIS::AmCyan::Trpl32) followed by an aadA cassette

(PpsbA::aadA::TrbcL (Chlamy) was constructed (Figure 6.28).

We used the usual protocol for plastid transformation in tobacco. Thus, either 7

days old protoplast derived colonies or three weeks old leaves from tobacco were

bombarded with gold particles covered with a pKCZ-AmCyan-aadA vector. Two

days after bombardment, protoplast derived colonies or leaf pieces were

transferred to selective RMOP medium (spectinomycin 500 mg/l) for 20 days.

Afterwards, medium was changed and plant material was incubated for

additional 20 days on RMOP medium (spectinomycin 500 mg/l). In order to

obtain transplastomic plants, several rounds of selection and regeneration were

carried out. Table 6.5 shows a summary of particle gun experiments in tobacco.

One experiment was performed with nine leafs, the other one with seven grids

containing protoplast derived colonies. Approximately, 5 putative lines were

gained per bombarded leaf and 2 putative lines were gained per each grid

containing protoplast derived colonies.

Table 6.5 Summary of particle gun plastid transformation experiments in Nicotiana tobacum

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Putative lines were further analysed by PCR, fluorescence microscopy and finally

by southern blotting. Some results are presented in figure 6.28, showing the

chloroplast transformation vector (pKCZ-AmCyan-aadA), pieces of leaves prior –

and after selection and amplification of the aadA gene by PCR from lines: 121, 151,

171, 191, 194 and 194-2. PCR amplification was positive (see arrow).

©

Figure 6.28 Plastid transformation in N. tabacum with the AmCyan gene. (a) Schematic presentations of the transformation vector containing AmCyan and aadA cassettes. Hatched boxes, homologous flanks, between trnR-ACG and trnN-GUU. Arrows indicate P1 and P2 for PCR amplification of the aadA gene. (b) Tobacco leaf pieces under selective conditions, two days after bombardment (RMOP-Spec 500 mg/l). (c) Three weeks after bombardment, showing a resistant shoot. (d) aadA amplifications from six lines independent lines. As positive control pKCZ-AmCyan-aadA vector was used, water was used as negative control and 1 Kb Plus was used as marker. Agarose TAE gel 1 %. Bars: 5 mm

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Positive lines were further characterized for AmCyan gene expression. All

chloroplasts in the analyzed lines expressed AmCyan (Figure 6.29). Fertile plants

were grown in the greenhouse to get seeds and sufficient plant material from T0

and T1 plants for Southern Blotting. Also, no difference in the phenotype between

tobacco wild type and AmCyan lines could be observed (Figure 6.30)

Southern Blot analysis allows us to check the correct integration of the AmCyan

gene in the plastome. Figure 6.30 displays the Southern Blot analysis and the

comparison of WT plants with one representative KAA line. Column 2 from

Southern blot shows the WT signal (5,701bp), in contrast, the analyzed lines in the

columns T1-T11 which display a DNA fragment signal from 8,561bp, indicating

the integration of AmCyan and aadA genes into the tobacco plastome. Line T8

shows a signal of circa 9,000 bp which can be explained by an intermolecular

recombination (Huang et al., 2002).

A weak wild type signal is observed in all positive lines, even after ten cycles

under selection (each cycle means regeneration induced during three weeks on

Figure 6.29 AmCyan accumulation in tobacco transplastomic chloroplast. (a) Stoma showing two guard cells under white light from a wild type plant. (b) Chlorophyll autofluorescence from a wild type plant. (c) Guard cells from wild type plant showing absence of AmCyan fluorescence. (d) Stoma showing two guard cells under white light from KAA194 line. (e) Chlorophyll autofluorescence from KAA194 line. (f) Guard cells from KAA194 line showing AmCyan fluorescence. Tobacco leaves were analyzed by confocal laser scanning microscopy. Bars: 10 µm

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83

RMOP containing 500 mg/l spectinomycin). This weak signal suggests that copies

from the wild type genome might still be present in all T1 analyzed lines.

Figure 6.30 Southern blot analysis of 10 transplastomic plants generated with a vector containing AmCyan fluorescence marker. (a) Schematic presentation of insertion sites in the tobacco plastome (ptDNA), hatched boxes: homologous flanks. Insertion occurred at the MunI site. The probe used for Southern blot analysis is indicated. (b) Eight weeks old wild type tobacco plant growing in the greenhouse (c) Eight weeks old tobacco KAA line growing in the greenhouse. (d) Southern blot analysis of 10 lines and one wild type control (wt). Total DNA was digested with NsiI and transferred to a nylon membrane. The blot was hybridized with the INSR probe (855 bp). Wild type resulted on a 5,701 bp fragment and transplastomic lines resulted on 8,561 bp fragment. Bars: 10 cm

To ensure the absence or presence of wild type plastome copies in those KAA

transplastomic lines, we performed reciprocal crosses of two independent

tranplastomic lines with wild type plants. This allows the screening of the progeny

for the presence or absence of a spectinomycin resistance gene in the plastome by

germination of seeds on selective medium. This method was proposed by Svab

and Maliga (1990) (Figure 6.31).

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84

Wild type and KAA lines containing an AmCyan and an aadA gene were

transferred to soil and grown to maturity. Seed pods were collected from selfed

plants and from reciprocal crosses. Surface sterilized seeds were germinated on B5

medium containing spectinomycin (500 mg/l). Self-pollinized transplastomic

plants and crosses with a transplastomic line as the maternal parent gave rise to

resistant (green) plants. Seeds collected from wild type plants yield antibiotic

sensitive (white) seedlings (data no shown) crosses with a wild type as the

maternal parent gave rise to sensitive plants. Importantly, pollen from

transplastomic lines does not transmit the transgenes (either aadA or AmCyan

genes) (Figure 6.31). Taken together, we could successfully introduce AmCyan, a

new fluorescence marker into chloroplast genome of Nicotiana tabacum.

Figure 6.31 Reciprocal crosses of transplastomic lines with wild type. (a) Schematic representation of reciprocal crosses between wild type and transplastomic plants. Pollen of transplastomic lines was used to pollinize wild-type (first cross), and pollen of wild-type plants was used to pollinize transplastomic lines (reciprocal crosses). (b) As a control, seedlings from self-crossed wild type tobacco plants were germinated on antibiotic-free medium. Seedlings develop normal. (c) Crosses between a transplastomic line as the maternal parent with pollen from the wild type tobacco plant as donor. Seedlings develop normal in the presence of spectinomycin, showing the maternal inheritance of the resistance cassette (green). (d) Crosses between wild type as the maternal parent with pollen from transplastomic line as donor. Maternal inheritance is shown by the antibiotic-sensitive (white) seedlings growing on spectinomycin-containing medium. Pollen of transplastomic plants does not transmit the transgene to the progeny.

Discussion

85

7 Discussion

7.1 Towards plastid transformation in Arabidopsis thaliana

Because Arabidopsis thaliana is a well characterized plant model organism

with thousands of available mutants, plastid transformation would be a helpful

tool for the elucidation of many aspects in plant biology. Thus, the establishment

of a reproducible plastid transformation method in Arabidopsis thaliana was

considered highly desirable for more than ten years (Sikdar et al., 1998; Maliga,

2004; Dhingra and Daniell, 2006).

From the biotechnological point of view, other alternatives as plant viral vector

systems represent a better option for transient expression of transgenes and large

scale production of biopharmaceutical proteins (Gleba et al., 2007). Nowadays,

plastid transformation is well established in tobacco and there it represents a tool

both, in basic and applied research (Koop et al., 2007; Maliga and Bock, 2011).

In this study, we focused on the most critical steps to achieve plastid

transformation in Arabidopsis thaliana and RCBr. To allow the generation of fertile

plants out of transformed cells, first, a reliable regeneration protocol must be

established. Then, for the transformation and specific introduction of genes into

plastids, an appropriate vector that ensures site specific integration and the

selection of the transformed cell is required. For selection, the vector often contains

an antibiotic resistance gene to detoxify antibiotics and a visual marker as GFP.

Finally, a plant transformation method such as particle gun or PEG mediated

transformation method must be established.

7.1.1 Regeneration protocols in Arabidopsis thaliana

For successful plastid transformation, a method which allows the

regeneration of fertile plants out of a single transformed cell is needed. Therefore,

in this study different protocols have been described using different cells and

tissue sources. On one hand tissues as cotyledons and seeds are used and on the

Discussion

86

other hand isolated plant cells without a cell wall (protoplasts) can be used for the

regeneration of fertile plants.

In Arabidopsis thaliana a major obstacle for plastid transformation was the lack of a

reproducible and efficient regeneration protocol (Acedo, 1986; Feldmann and

Marks, 1986). In this study we established reliable tissue and cell culture

procedures allowing high regeneration efficiencies.

Tissue culture

Most regeneration protocols using cotyledons or seeds in Arabidopsis

thaliana follow a two-step procedure. First, explants are dedifferentiated with the

help of a callus induction medium (intermediate auxin : cytokinin ratio) and

subsequently, the callus is transferred to a shoot induction medium (low auxin :

high cytokinin) (Valvekens et al., 1988).

Regeneration efficiencies in Arabidopsis thaliana also depend on ecotype and tissue

type. Between all the analysed ecotypes, C24 has one of the highest organogenesis

and embryogenesis capacities (Schmidt and Willmitzer, 1988; Chaudhury and

Signer, 1989; O'Neill and Mathias, 1993; Barghchi et al., 1994). Recently our group

confirmed these finding in Arabidopsis thaliana ecotype C24 (Luo and Koop, 1997;

Dovzhenko et al., 2003).

On the basis of these results, we used several explants of the ecotype C24 in this

study. Furthermore, by the combination of several previously reported protocols,

the regeneration rate was improved.

The use of cotyledons as explants has been previously described. In this thesis, a

callus induction medium was applied according to Zhao et al., (2002) and a

regeneration medium as described by Luo et al., (1999). Shoots could be

regenerated with efficiencies of up to 85 % using 6 days old cotyledons. This is

almost 4 times more than previously reported by Zhao et al., (2002).

Successful establishment of a regeneration protocol also depends on the age and

the origin of the explants. A reduction in the regeneration capacity was observed

Discussion

87

when two weeks old leaves were used (personal communication with Salar

Torabi). Other explant sources have been used in the regeneration of shoots in

Arabidopsis thaliana including hypocotyls, leaves (Schmidt and Willmitzer, 1988),

roots (Valvekens et al., 1988) and seeds (Negrutiu et al., 1975).

Regeneration of plants using seeds as explants for the induction of organogenesis

or embryogenesis has been reported by several groups (Negrutiu et al., 1975;

Huang and Yeoman, 1984). However, high regeneration efficiency was not

achieved by those groups. Thus, we also tried to improve this type of regeneration

protocol. To allow the selection of transformed plastomes in the presence of

selective agents, we have chosen a regeneration method with a long period of

callus induction. For this reason, seeds were first incubated in a callus induction

media for four weeks. With a combination of the callus induction medium

PG20KN and the shoot induction medium SRAKN, we have reached efficiencies

of up to 97 %. If each shoot per explant is counted independently, we could reach

an efficiency of 500 to 700 % (Figure 6.4).

Atta et al., (2009), presented some evidence that the origins of shoot regeneration

are pericycle cells. They demonstrated that xylem pericycle is much more

pluripotent than previously thought. In this study, when seed derived calli were

transferred to regeneration medium shoots were formed either from roots or from

hypocotyls. This data supports previous findings by Atta et al., (2009) because

xylem pericycle cells can be found in hypocotyls and roots.

Protoplast culture

Plastid transformation efficiencies using PEG-mediated DNA transfer or the

particle gun method have almost the same transformation efficiencies in tobacco

(Kofer et al., 1998). This makes protoplasts a good alternative for plastid

transformation. Successful protoplast cultivation depends on the following factors:

the correct environmental conditions, source of protoplasts, protoplast density,

media composition, osmotic pressure, light, pH and temperature conditions

(Dovzhenko et al., 1998).

Discussion

88

In this work, an efficient regeneration protocol for protoplast culture in Arabidopsis

thaliana was established. Embedding protoplasts in alginate has the advantage that

it allows better the cell division and thus the regeneration of fertile plants out of

leaf protoplasts (Damm and Willmitzer, 1988). Plating efficiencies (number of

protoplast divisions per embedded protoplasts) of up to 16 % could be reached

after embedding of Arabidopsis thaliana leaf protoplasts in thin alginate layers (Luo

and Koop, 1997).

Previously, it was shown that fertile plants could also be regenerated from

different sources like protoplasts from suspension cultures (Ford, 1990), roots

(Mathur et al., 1995), leaves (Luo and Koop, 1997) and cotyledons (Dovzhenko et

al., 2003).

As a base for our experiments, we used a protocol proposed by Dovzhenko et al.,

(2003) with some modifications that provided plating efficiencies between 40 to

60 %. An important modification of the method by Dovzhenko et al. was the

replacement of the Dicamba (auxin) phytohormone with a combination of the

auxin NAA and the cytokinin 2ip in the protoplast culture medium (PCA).

Another important change was the addition of the antibiotic carbenicillin to avoid

contamination of protoplast cultures with bacteria. Once protoplast derived

colonies were obtained, grids were transferred to a shoot regeneration medium

with Kinetin and NAA as phytohormones (SRAKN).

Regeneration in SRAKN medium was highly efficient and more than 150 shoots

per grid were obtained. Shoots appeared on SRAKN medium after just 7 days of

culture (Figure 6.7).

To allow the segregation of transformed chloroplasts, a regeneration medium with

a callus phase was established. Therefore, SRA2N medium was supplemented

with 2ip and NAA as phytohormones which allow a long callus phase before

shoot formation.

Protoplast-derived calli contain a lower number of plastids compared to leaves

(Thomas and Rose, 1983). This could facilitate to a faster selection of transformed

Discussion

89

chloroplasts because the starting ratio of transformed plastids to untransformed

plastids is higher from the beginning on. Thus, the transformed plastomes will

have a better opportunity of being transmitted to daughter cells (Huang et al.,

2002).

7.1.2 Species specific vectors in Arabidopsis thaliana

Plastid transformation not only requires a reliable regeneration method, it

also needs species specific vectors with appropriate regulatory elements, selective

markers and specific recombination sites.

Construction of species specific vectors

Plastid transformation follows the principles of homologous recombination

(Carrer and Maliga, 1995). The transgene has to be flanked at each site by plastome

sequences ensuring the integration of the transgene into a specific region of the

plastome (Figure 3.4) (Svab et al., 1990).

Plastid transformation has been achieved using heterologous sequences to allow

the integration of transgene in plastomes. Nevertheless, a lack of complete

homology may reduce the transformation efficiency, for example, when petunia

flanking sequences are used to transform tobacco plants (DeGray et al., 2001).

Thus, the use of species-specific vectors for each crop is suggested (Table 7.1).

Selection of integration sites and size of homologous flanks could be an important

factor to achieve transplastomic plants. In this work we have used three

homologous flanks to the Arabidopsis thaliana plastome for the insertion of

transgenes. These three regions are between rps12/7-trnV (Zoubenko et al., 1994);

trnfM-trnG (Ruf et al., 2001) and rbcL-accD genes (Svab and Maliga, 1993).

In summary, insertion sites used in this study for plastid transformation vectors in

Arabidopsis thaliana have been previously reported in several plants as shown in

Table 7.1.

Discussion

90

Figure 7.1 Schematic representation of transcription-initiation sites in the promoters of the rrn operon in spinach (So), Arabidopsis (At), and tobacco (Nt). Transcription-initiation sites are marked with horizontal arrows PEP and nuclear-encoded plastid RNA polymerase promoters are marked with open and filled circles, respectively. Figure from Sriraman et al., 1998.

Regulatory elements in plastid transformation

Regulation of plastid expression in higher plants is similar to that in

eubacteria. The plastome encodes the RNA polymerase (PEP) and RNA

polymerases (NEP) as RPOTp and RPOTpm are imported into plastids from the

nucleus (Allison, 2000).

In plastid transformation the most common promoter used is the rrn promoter

(Prrn) of the 16S plastid rRNA operon. This Prrn promoter is regulated during

development and its operon can be transcribed by both, PEP and NEP

polymerases. Regulation of the rrn operon is complicated as this operon has three

promoters: P1, PC and P2. Tobacco chloroplasts lack the factor required for PC

recognition, however other species like spinach and Arabodopsis thaliana are able to

initiate transcription from PC sites. In Arabidopsis thaliana the RPOTmp transcribes

the rrn operon using the PC promoter only in early developmental stages

(Courtois et al., 2007).

In contrast to this evidence, one of the initial findings in Arabidopsis thaliana

transplastomic plants was that Prrn from tobacco is functional in Arabidopsis

thaliana plants even if it lacks the PC region (Sriraman et al., 1998). This fact can be

explained by the presence of the P1 promoter in tobacco. Figure 7.1 shows the

structure of the rrn operon promoters.

Due to the fact that the tobacco Prrn promoter has been proven to function in

Arabidopsis thaliana, we decided to use this promoter in our plastid transformation

vectors.

Discussion

91

The psbA promoter was another promoter we chose. The psbA gene encodes the

D1 protein of photosystem II and its promoter is often used in plastid

transformation (Staub and Maliga, 1993; Tangphatsornruang et al., 2010).

Post-transcriptional RNA levels are also tightly regulated in chloroplasts (Stern et

al., 2010). Stem-loop secondary structures in the 5´ UTR of the mRNA are

important for the translation and the stability of the mRNA (Eibl et al., 1999;

Monde et al., 2000b). For these reasons, a suitable 5 ÚTR region which includes a

ribosomal binding site (RBS) is important for successful plastid transformation.

Usually, a 5ÚTR of a highly expressed plastid gene like atpB, psbA or rbcL is used

(Staub and Maliga, 1993).

It has been shown that a 5ÚTR of the gene 10 from the bacteriophage T7 (T7g10)

increases the translation rate and thus the accumulation of proteins (Kuroda and

Maliga, 2001). Several groups have shown that extensions or modifications of the

N-terminus of the transgene protein also increase the expression level of proteins

(Kuroda and Maliga, 2001; Ye et al., 2001; Apel et al., 2010).

Another important element for transcript stability is the 3ÚTR. Several 3ÚTRs

have been tested. Eibl et al., (1999) found that the 3ÚTR from the rpl32 gene could

increase the amount of uidA transcripts up to threefold compared to the psbA

3ÚTR. However, GUS activity was not significantly influenced in transplastomic

tobacco plants. Stability of transcripts has also been confirmed using different

3ÚTR sequences from rbcL, psbA, petD, rpoA and E. coli rrnB genes and gfp as

reporter gene (Tangphatsornruang et al., 2010). In summary, regulatory elements

are important for plastid transformation and should be considered carefully. Table

7.1 gives an overview of the regulatory elements used in this study, their

references and a comparison with other plants.

Discussion

92

Combination of visual and selection markers

The combination of a visual with a selection marker allows a simpler

selection of transformed cells. In this study, vectors containing selection markers

such as aadA and aphA6 outside the homologous flanks and visual markers inside

the flanks were constructed.

This strategy is based on the generation of marker free transplastomic plants

proposed by Klaus et al., in our group (Klaus et al., 2004). Klaus et al., observed that

recombination via a single flank often occurs; this event leads to the formation of

vector co-integrates in the plastome. This co-integrate is resolved through a

secondary recombination event. Once selection pressure is removed, a secondary

Table 7.1 Plastid transformation in higher plants*

Discussion

93

Figure 7.2 Marker free plastid transformation. Plastid transformation vector with a selection marker (SM) outside of the homologous flanks (dark boxes). Recombination can occur via either left of right flanks resulting in co-integration of the vector in the plastome. After several recombination events and without selection pressure the marker gene is lost. Finally only the gen of interest (GOI) is stable integrated or the wild type plastome could be obtained. Uses of a visual marker as GOI can favor the selection of transplastomic lines.

recombination either leads to plastomes identical to those of the target lines or to

marker free plastomes containing the gene of interest (Figure 7.2). Klaus et al. used

a system in which pigment-deficient tobacco knockout plants (ΔpetA or ΔrpoA)

were used as a target. The transformation vector carried a gene which encodes a

protein involved in photosynthesis; this restored the green pigmentation and

introduced the gene of interest with the removal of the selective marker.

Homoplastomic sectors in plants were identified by the green color; this strategy

reduces significantly the time required to obtain homoplastomic plants and avoids

problems with contamination of transgenes (Klaus et al., 2003; 2004).

7.1.3 Particle gun-mediated plastid transformation

Cotyledons and seed derived calli as target for plastid transformation

In the beginning of this study, cotyledons (6 days old) of Arabidopsis thaliana

were used for plastid transformation experiments. Petri dishes containing 150

cotyledons were bombarded with appropriate plastid transformation vectors. Due

to the fact, that 6 days old cotyledon preparations and their subsequent transfer to

Discussion

94

selection medium was a time consuming work and all nine particle gun

experiments failed, we decided to simplify our method by the use of seed derived

calli.

After the improvement of the regeneration protocol for seed derived calli, the

appropriate concentration for the selective reagents as bialaphos and PPT was

determined. Therefore, seed derived calli were bombarded with pBA-GFPbar or

pC24-GFPbar vectors (Table 7.1). Seed derived calli were further grown in callus

induction media containing different concentrations of bialaphos or PPT (data

none shown). The bar gene encodes for resistance to the herbicides bialaphos and

PPT (Lutz et al., 2001). In media containing B5 vitamins (PG1KD) the generation of

shoots was inhibited after the addition of 1.5 mg/l bialaphos. Interestingly, in the

presence of cocktail 20 almost twice the concentration of bialaphos (3 mg/l) was

needed. Cocktail 20 contains a high concentration of nitrogen compounds which

may influence herbicide activity. At least 170 Petri dishes containing seed derived

calli (one or four weeks old) were bombarded. However, under selective

conditions, no resistant line was obtained and no GFP fluorescence could be

detected.

To test that the biolistic gun experiment works in principal, transient experiments

using a nuclear vector containing the uidA gene (data not shown) were performed.

The histochemical detection of GUS in cotyledons and seed derived calli

demonstrated that the chosen biolistic gun conditions were suitable for DNA

delivery into the cells and that those conditions can be used for Arabidopsis thaliana

plastid transformation.

Protoplast derived colonies as target for transformation

The use of protoplast culture for plastid transformation experiments is not

limited to the PEG-mediated plastid transformation method. Protoplast derived

colonies can be used as target for particle gun-mediated plastid transformation. In

1998 Dovzhenko et al., described a simplified method for the handling of

protoplasts and the embedding of the cells into alginate which enhances cell

Discussion

95

divisions and thus the formation of protoplast derived colonies (Dovzhenko et al.,

1998). Use of protoplast derived colonies as target for plastid transformation in

tobacco reduced the handling effort by 60 % compared to the traditional use of

leaves as target material (Huang et al., 2002).

In this study, after the improvement of protoplast culture, more than 350 petri

dishes containing protoplast derived colonies were bombarded with DNA coated

particles. Several vectors were used (Table 7.1). The first set of experiments was

performed with vectors containing DsRed fluorescence marker and the genes aadA

and aphA6 to confer resistance to spectinomycin and kanamycin antibiotics

95olyplotively. From this experiment, 12 resistant calli were obtained. However,

under more strict selection conditions all calli and shoots bleached. DsRed

fluorescence analysis was difficult because the high background due to the auto

fluorescence of wounded and highly stressed cells. Using this method no positive

lines were detected.

According to a publication by Jach et al., (2001) tobacco plants could be

successfully transformed with DsRed-Chl via Agrobacterium tumefaciens. DsRed

gene was integrated into the nucleus and the DsRed protein was imported into

chloroplasts with the help of a signal peptide. However, DsRed could only be

observed in fully developed plant, and not in calli (Jach et al., 2001). This could be

the reason why we could not detect DsRed in our calli.

In a second set of experiments, the fluorescence marker AmCyan was combined

with the aadA gene which confers spectinomycin resistance. In these experiments

89 putative lines were obtained. One advantage of AmCyan is that it is highly

resistant to photobleaching and it has a relatively high brightness level when

compared to CFP (Cyan Fluorescent Protein) from Aequorea (Day and Davidson,

2009). Recently, in our group, Torabi (2009) achieved plastid transformation in

Brassica napus using the AmCyan fluorescence marker. As reported by Torabi

(2009), slight blue fluorescence was observed in the cell wall of wild type

Arabidopsis thaliana (Torabi, 2009). Buschmann et al., have shown that the blue

green fluorescence (440 nm) derived from ferulic acid (a hydroxycinnamin acid)

Discussion

96

bound to the cell wall (Buschmann et al., 2000). Although the low background

made the analysis more complicated, we used this marker together with a

fluorescence microscope. As a positive control, we took the transgenic Arabidopsis

thaliana line pt-ck CS16265 (Nelson et al., 2007), expressing AmCyan in

chloroplasts (data no shown). This transgenic pt-ck CS16265 was nuclear

transformed and AmCyan protein was imported into chloroplasts with the help

from a peptide signal. In comparison with this line, all 89 putative lines were

negative for AmCyan fluorescence.

One reason for the failure of the selection of transformants is probably the very

fast shoot formation. Shoot generation started at the 5th day on SRAKN medium.

Once shoots are formed, antibiotics cannot penetrate the inner cells of this shoots,

which lead to an insufficient selection. The other hypothesis is that only some

plastomes were transformed and that the level was not high enough to detect

them by fluorescence microscopy or PCR.

Plastid transformation requires multiple rounds of selection to eliminate wild-type

plastomes and to achieve a homoplastomic state. In this study, a regeneration

medium with a long callus phase (SRA2N) was used, this allows multiple cycles of

selection and regeneration. Additionally, vectors containing gfpbar genes or just

one selection marker as the aadA or the nptII genes were used. The nptII gene

includes the first 14 amino acids of the GFP protein, which improved the selection

of transplastomic plants in tobacco (Scharff and Koop, 2007). Despite of all these

strategies, all putative lines were negative for transgenes and they bleached after

prolonged selection (Table 7.1).

7.1.4 PEG-mediated plastid transformation

An alternative for particle gun experiments is PEG-mediated plastid

transformation. One advantage of the PEG method over the particle gun method is

that for PEG no expensive equipment is required and in Nicotiana tabacum the

PEG-mediated plastid transformation method is equally efficient as the biolistic

Discussion

97

gun method (Kofer et al., 1998). For PEG-mediated plastid transformation, isolated

protoplasts are treated with PEG in the presence of DNA. Successful PEG-

mediated plastid transformation needs a regeneration protocol which allows the

regeneration of fertile plants from protoplasts. Our group could stably transform

chloroplasts in Nicotiana tabacum using PEG, for the first time in 1993 (Golds et al.,

1993) and shortly afterwards O´Neill et al., were able to repeat the same in

Nicotiana plumbaginifolia (O'Neill et al., 1993). PEG-mediated plastid transformation

was also applied in other plants as Solanum lycopersicum (tomato) (Nugent et al.,

2005b), Brassica oleracea var. botrytis (cauliflower) (Nugent et al., 2005a) and Lactuca

sativa (lettuce) (Lelivelt et al., 2005). However, more than 15 years later, the precise

mechanism of how DNA is transported through the double membrane of

chloroplasts is still unknown (Spörlein et al., 1991).

We performed at least 20 PEG-mediated plastid transformation experiments and

in each experiment 1 x 106 protoplasts were used. Although several different

plastid transformation vectors were constructed and used, no transplastomic plant

was obtained.

To show that PEG-mediated transformation works in principle and that plants can

be regenerated after the PEG treatment, GFP was expressed transiently in the

cytoplasm of nuclear transformed protoplasts (Figure 6.23) and plants were

regenerated. With this method we also tested different PEG concentrations (20 %

and 40 %). We could also regenerate plants out the treated protoplasts confirming

that the method allows the transformation and regeneration of plants out of

treated protoplasts.

7.2 Towards plastid transformation in Rapid-Cycling

Brassica rapa

In parallel to Arabidopsis thaliana tissue culture, we have worked on the

establishment of a regeneration protocol for Rapid-Cycling Brassica rapa (Figure

6.25). Although those Rapid-Cycling Brassica rapa (RCBr) plants have been recently

proposed as a new model organism (Williams and Hill, 1986), this model lacks a

Discussion

98

reproducible tissue culture regeneration protocol. Up to the date, just two

protocols using cotyledons as explants have been described (Teo et al., 1997;

Cogbill et al., 2010). We intended to reproduce their findings, but we could not

obtain shoots although using both protocols in RCBr C1 and B3 plants. Then, we

modified the explant preparation protocol by Jonoubi et al., for Brassica napus

(Jonoubi et al., 2005). Hypocotyls were cut at their sagittal axis to expose the

vascular tissues. This modification allowed regeneration efficiencies of 50 to 80 %

in C1 and 20 to 50 % in B3 lines. However, the high regeneration efficiencies are

very sensitive to environmental changes (personal communication with Peter

Wehmann). Thus, factors as explant sources and origin of seeds need to be

considered for tissue culture.

In this study, at least 10 particle gun-mediated plastid transformation experiments

with a species specific vector for RCBr (Fig 6.25) were performed. Although one

putative callus was obtained, it bleached after strict selection conditions.

In general, plastid transformation in crops is not as efficient as in tobacco (Table

7.1). In tobacco at least two or more transplastomic plants are obtained per

bombardment. However, in crop plants as Oriza sativa or Glycine max more than 20

particle gun experiments are needed to acquire transplastomic plants (Koop et al.,

2007). For this reason, more experiments in RCBr may be necessary to achieve

plastid transformation.

7.3 Nicotiana tabacum, a positive control in plastid

transformation

In this study Nicotiana tabacum was used as a positive control for plastid

transformation. One the one hand, Nicotiana tabacum was taken to demonstrate

that the AmCyan gene can be used as a new visual marker and on the other hand,

it allowed the test of regulatory elements used in Arabidopsis thaliana plastid

transformation vectors. Nicotiana tabacum is the first higher plant in which plastids

could be transformed by the particle gun method (Svab et al., 1990). One

advantage of tobacco is its high regeneration capacity. Tobacco is still a model

Discussion

99

plant for cell and tissue culture research. Protoplast regeneration from Nicotiana

tabacum was described for the first time in 1971 (Nagata and Takebe, 1971). Here,

following a method of Dovzhenko et al., high platting efficiencies could be

achieved allowing shoot formation in less than two weeks (Dovzhenko et al., 1998).

Leaves and protoplast derived colonies were bombarded with the pKAA vector

containing aadA and AmCyan expression cassettes (Figure 6.28). The pKAA

vector contains flanking regions from the pKCZ vector for the insertion of the

transgene between the trnN-trnR genes from Nicotiana tabacum (Zou et al., 2003).

First putative lines were obtained after four weeks under selection conditions.

Although AmCyan fluorescence could not be detected using a fluorescence

stereoscope, blue fluorescence could clearly been observed using a laser scanning

microscope (Figure 6.29). T0 and T1 lines expressing AmCyan in chloroplasts of

Nicotiana tabacum were obtained. Transformation efficiencies were comparable to

those obtained by using other selection markers in Nicotiana tabacum (Huang et al.,

2002).

Further analysis of these lines by southern blotting have shown a very faint band

suggesting that wild-type plastome sequences could still be present in these lines,

even after seven cycles of regeneration. One explanation for this faint signal could

be the presence of plastid DNA sequences in the nuclear genome (Ayliffe et al.,

1989; Huang et al., 2002). This hypothesis is supported by the fact that reciprocal

crosses and screening of the progeny allowed the confirmation of the

homoplasmic status of the analyzed lines (Figure 6.31).

In summary, these results show that the reef coral fluorescent protein AmCyan

can be used as visual marker in plastid transformation of Nicotiana tabacum. The

results also clearly show that all the expression cassettes used were fully

functional.

Discussion

100

7.3.1 Plastid transformation in Arabidopsis thaliana and

Nicotiana tabacum

For plastid transformation experiments in Arabidopsis thaliana several

experimental approaches could be established: 1. A highly reliable regeneration

protocol was developed. 2. Plant specific vectors for plastid transformation were

constructed. 3. Vectors with different fluorescence and selection markers were

tested and 4. Particle gun- and PEG-mediated plastid transformation methods

were performed. Notwithstanding, after all this attempts, no plastid transformant

could be obtained in Arabidopsis thaliana. More than 10 years have passed since the

transformation of Arabidopsis thaliana plastids was reported (Sikdar et al., 1998) and

several groups have tried stable transformation of plastids in Arabidopsis thaliana,

but no success has been reported so far. It is still not clear where the bottleneck is.

Until now, Nicotiana tabacum is still the best plant model organism where plastid

transformation has been achieved (Koop et al., 2007; Maliga and Bock, 2011).

Recently, evidence for microhomology and nonhomologous end joining (NHEJ)

repair mechanisms of double strand breaks (DSB) was described in chloroplasts of

Arabidopsis thaliana (Kwon et al., 2010). Interestingly, in Nicotiana tabacum

chloroplasts lack the capacity to repair DSB in their plastomes by the NHEJ

mechanism (Kohl and Bock, 2009). Chloroplasts are constantly exposed to reactive

oxygen species generated during photosynthesis which may cause permanent

DNA damage (Maréchal and Brisson, 2010).

The most common form of DNA damage is a double strand break (DSB). In plants

mechanisms of DSB repair includes homologous recombination (HR),

microhomology and NHEJ. According to Kohl and Bock, (Kohl and Bock, 2009) it

has been shown with the help of an insertion sequences (IS) of Escherichia coli that

this IS element is mobilized inside the chloroplast. These results suggest that

plastids of Nicotiana tabacum cannot repair DSB through NHEJ which is important

for their implications in genome stability. Understanding of the mechanisms of

DSB repair may help to understand the underlying mechanisms of plastid

transformation in Arabidopsis thaliana.

Discussion

101

7.4 Future directions in plastid transformation of


Several groups attribute failures of plastid transformation methods in

Arabidopsis thaliana to the homologous recombination machinery (Sikdar et al.,

1998). Although this argument cannot completely be discarded; recent studies

have shown that Arabidopsis thaliana has an efficient homologous recombination

system (Rowan et al., 2010).

Despite the highly reactive forms of oxygen in plastids, plastomes are still as

conservative genomes as nuclear or mitochondrial genomes (Kwon et al., 2010),

meaning that efficient DNA repair mechanisms must exist in plastids. Despite the

need of a better understanding of homologous recombination in plastids, there is

little knowledge on the mechanisms and enzymes involved in this process. In

Arabidopsis thaliana some proteins involved in plastid DNA replication,

recombination and repair (RRR) have been identified. These proteins include

RecA-like recombinases and two families of plant specific single-stranded DNA

(ssDNA)-binding proteins: the organelle ssDNA-binding proteins (OSBs) and the

Whirly proteins (Marechal et al., 2009; Maréchal and Brisson, 2010).

In E. coli the rec-A protein is well characterized. It is involved in the reduction of

mutations and in the repair of DNA by homologous recombination. Homologues

of recA have been identified in algae and in the nuclear genomes of several plants,

including Arabidopsis thaliana (Cerutti et al., 1992). It has also been shown that the

absence of recA gene induces aberrant plastomes in Arabidopsis thaliana.

Interestingly, the variegated phenotype of RecA mutants could be observed only

after four generations (Rowan et al., 2010).

Transgenic Arabidopsis thaliana plants overexpressing a RecA gene could increase

the success of homologous recombination and thus the stable transformation of

plastids after bombardment with DNA covered particles. This strategy has already

been tested in Nicotiana tabacum with positive results (Liu, 2005).

In Arabidopsis thaliana it has been shown that plastid DNA copy number remains

constant during leaf development (Li et al., 2006; Zoschke et al., 2007). This means,

Discussion

102

that each daughter cell receives in theory an equal number of plastids and that the

plastid genomes are inherited strictly randomly. Plastid transformation requires

several regeneration cycles under selective conditions to allow the segregation of

transformed plastomes. As suggested by Bogorad (2000), the reduction of the

number of chloroplasts per cell may also favour the selection of transformed

plastomes.

Reduced numbers of chloroplasts are observed in mutants or transgenic plants

with macrochloroplasts in Arabidopsis thaliana. Through antisense mechanisms that

inhibit the expression of either the AtFtsZ1-1 or the AfFtsZ2-1 genes in Arabidopsis

thaliana, the number of 100 chloroplasts has been reduced to one macrochloroplast

(MCP) per leaf cell (Osteryoung et al., 1998). Plants containing MCP were obtained

by the overexpression of the AtMinE1 or the AtMinD1 in the nucleus (Colletti et al.,

2000; Maple et al., 2002; Aldridge et al., 2005; Glynn et al., 2007). The use of

transgenic plants containing macrochloroplasts in plastid transformation has been

further explored in Nicotiana tabacum plants (Chikkala, 2009). The plastid division

gene MinD from cauliflower was overexpressed in tobacco plants and afterwards

transgenic plants containing macrochloroplasts were used for plastid

transformation experiments. Interestingly transformation efficiencies were not

increased. However, only one regeneration cycle was necessary to obtain

homoplasmy (Chikkala, 2009). This significant reduction of selection cycles would

make transgenic plants containing MCP a good alternative for plastid

transformation in Arabidopsis thaliana.

Another possibility to improve plastid transformation could be the use of

tetraploid Arabidopsis thaliana plants for particle gun experiments. In this regard, it

is interesting to note that most plants which could successfully plastid

transformed are polyploidy. Most prominent is Nicotiana tabacum with four copies

of the genome. In contrast, Arabidopsis thaliana has just two copies of its genome.

Thus, it would be an interesting experiment to try plastid transformation of

polyploidy Arabidopsis thaliana.

Abbreviations

103

8 Abbreviations

2,4-D 2,4-Dichlorophenoxy acetic acid

aadA 3´Aminoglycoside-adenyltransferase

aphA6 3´Aminoglycoside phosphotransferase type VI

ATP Adenosin 5´-triphosphat

B5 Gamborg´s medium

BAP Benzylaminopurine

BD Binding domain

cm Centimeter

CTAB Cetyl trimethyl ammonium bromide

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

eGFP Enhanced green fluorescent protein

EM Electron microscopy

EtBr Ethidium bromide

EtOH Ethanol

GFP Green fluorescent protein

h Hour

IAA 3-Indole acetic acid

IBA Indole butyric acid

IR Inverted repeat region

KoAc Potassium acetate

mRNA messenger Ribonucleic acid

MS Murashige and Skoog medium

NAA Naphthalene acetic acid

NEP Nuclear-encoded RNA polymerase

nptII Neomycin phosphotransferase II

ºC Degree Celsius

p.a Analytical reagent quality

pat Phosphinothricin acetyl transferase

PCR Polymerase chain reaction

PEG Polyethylene glycol

PEP Plastid-encoded RNA polymerase

pp Protoplasts

PPT Phosphinothricin

Prrn Plastid 16 S r RNA promoter

RCBr Rapid-Cycling Brassica rapa

RNA Ribonucleic acid

rpm Revolutions per minute

s Seconds

SD Shine-Dalgarno sequence

SDS Sodium dodecyl sulfate

Spec spectinomycin

UTR Untranslated region

UV Ultraviolet light

V volt

wt wild-type

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104

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Curriculum vitae

122

10 Curriculum vitae

NAME: Areli Herrera Díaz

DATE OF BIRTH: November 16th, 1979

PLACE OF BIRTH Tampico, Tamaulipas, Mexico

MARITAL STATUS: Single

NATIONALITY: Mexican

EDUCATION:

10.2006-04.2011 PhD thesis at the Ludwig-Maximilians-University of Munich, Germany. Project: Regeneration and plastid transformation approaches in Arabidopsis thaliana and Rapid-Cycling Brassica rapa. Director: Prof. Dr. Hans-Ulrich Koop. PhD scholarship: DAAD (Deutscher Akademischer Austausch Dienst)

04.2006-09.2006 German course

08.2003-12.2005 Master Degree in Molecular Biology at the Instituto Potosino de Investigación Científica y Tecnológica, S.L.P, Mexico. Project: Nuclear and Plastid transformation of vegetal systems with antimicrobial genes. Director: Dr. Ángel G. Alpuche Solís.

08.1997-03.2003 Chemist Bacteriologist Parasitologist, at the Biological Sciences Faculty, Universidad Autónoma de Nuevo León. Monterrey, N.L. Mexico.

08.1994-07.1997 Clinic laboratorist technician and High School at the Technical Medical,Universidad Autónoma de Nuevo León. Monterrey, N.L. Mexico.

PUBLICATIONS:

Noyola, D.E., Alpuche-Solís, A.G., Herrera-Díaz, A., Soria-Guerra, R.E., Sánchez-Alvarado, J., López-Revilla, R.H. "Human metapneumovirus infections in Mexico: Epidemiological and clinical characteristics." Journal of Medical Microbiology. 2005; 54: 969-974

Rosales-Mendoza, S., Alpuche-Solís, A., Soria-Guerra, R., Moreno-Fierros, L., Martínez-González, L., Herrera-Díaz, A., Korban, S.S. "Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin and its assembly as a functional oligomer in transplastomic tobacco plants". Plant Journal. 2009; 57: 45-54.

Rosales-Mendoza, S., Soria-Guerra, R.E., Moreno-Fierros, L., Govea-Alonso, D.O., Herrera-Díaz A., Schuyler S. K., Alpuche Solís A.G. “Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants” Plant Cell Report. 2011, in press

Herrera Díaz, A., and Koop, H.U. Nicotiana tabacum–PEG-mediated plastid transformation. Methods and Protocols. 2011, in press

Acknowledgements

123

11 Acknowledgements

I am deeply grateful to Prof. Dr. Hans-Ulrich Koop for accepting me in his

group, for the interesting project and of course thank you Ulrich, for the friendly

and constructive discussions during my research works.

I am thankful to Prof. Dr. Jörg Nickelsen for reviewing this work.

I would also like to thank Prof. Dr. Angelika Böttger, Prof. Dr. Elisabeth Weiss,

Prof. Dr. Gerhard Wanner and Prof. Dr. Ute C. Vothknecht for reading this

manuscript.

Special thank for Dr. Stefan Lamla for his assistance during the writing process

and to Dr. Carlos Acevedo and M.Sc. Laura Verastegui for the proof-reading.

I am deeply grateful for Stefan Kirchner´s excellent technical assistance, his

friendly cooperation and his willingness to help. Thank to Dr. Sophie Bundtzen

for the scientific discussions and her friendship. Thanks to all my dear colleges

Marika Hopper, Salar Torabi, Peter Wehmann, Caro Adams and Florian Mayer for

the productive time in the lab and the nice after work activities. Thanks to Dr.

Christian Stelljes for all the lab tips during my research, his friendly help and for

his computer assistance.

I am thankful to the gardener team at the institute, especially to Ulrich Wißnet and

Ariel Rosenberger for their care taking on my plants.

Heartfelt thanks goes to DAAD (Deutscher Akademischer Austausch Dienst) for

their financial support.

Thanks to my host mother Karin Burges for the nice time in Eichenau.

I need to say thank you to my friends in Mexico for their company in the distance,

Diana Caballero, Leonardo Chávez, Alma Rincon, Cecilia Silva, Aurora Londoño,

Sergio Rosales, Pedro Ojeda and Maribel Hernández.

Of course I am grateful to my friends in Munich for the nice time here, Jennifer

Borelly, Sergio Prieto, Carlos Acevedo, Ernesto Márquez, Saura de Koop,

Montserrat Miramontes, Rebeca de Schupp, Julieta de Dürr, Berenice Malvido,

Eduardo Aguilar and Luis Tay, thank you all for the pretty time in Germany.

My deepest gratitude goes to my father Juan Herrera Ríos, my mother Lupita

Palomares Villalobos, my brother Erick Alejandro Herrera Palomares and to my

family for all the years of support, patience and love (Gracias por todos estos años

de apoyo, paciencia y cariño). Lastly, my special thanks to Stefan for his support,

understanding and the beautiful time together.

Erklärung

124

12 Erklärung

Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir

selbständig und ohne unerlaubte Hilfe angefertigt ist. Ich habe weder an anderer

Stelle versucht eine Dissertation oder Teile einer solchen einzureichen bzw. einer

Prüfungskommission vorzulegen, noch eine Doktorprüfung zu absolvieren.

München, den 28.04.2011

Regeneration and plastid transformation approaches in Arabidopsis thaliana and Rapid-Cycling

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